Cancer antigen helper peptide

ABSTRACT

The present invention relates to a WT1 peptide which has an amino acid sequence consisting of contiguous amino acids derived from a WT1 protein and induces WT1-specific helper T cells by binding to an MHC class II molecule, a pharmaceutical composition comprising them and the like.

The present application is a continuation of U.S. application Ser. No.15/831,484, filed Dec. 5, 2017, which is a continuation of U.S.application Ser. No. 14/994,801, filed Jan. 13, 2016, which is adivisional of U.S. application Ser. No. 13/265,806, filed Oct. 21, 2011(now U.S. Pat. No. 9,266,932), which is a National Stage entry ofPCT/JP2010/057149, filed Apr. 22, 2010, and claims priority to JapanesePatent Application No. 2009-105286, filed Apr. 23, 2009, the disclosuresall of which are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 1, 2015, isnamed 05273_0135_SL.txt and is 58,313 bytes in size.

TECHNICAL FIELD

The present invention relates to WT1 helper peptide, a polynucleotideencoding the peptide, WT1-specific helper T cells induced by thepeptide, a pharmaceutical composition for treating/preventing cancercomprising them and the like.

BACKGROUND ART

The WT1 gene (Wilms' tumor 1 gene) is a gene identified as a causativegene of a Wilms' tumor which is a kidney cancer in childhood (Non-PatentDocuments 1 and 2), and is a transcription factor having a zinc fingerstructure. At first, the WT1 gene was considered to be a cancersuppressor gene. However, subsequent investigation showed that the abovegene rather serves as a cancer gene in hematopoietic organ tumors andsolid cancers (Non-Patent Documents 3 to 6).

Since the WT1 gene is highly expressed in many malignant tumors, a WT1gene product which is a self-protein having no mutation has beenverified for existence or non-existence of immunogenicity in vivo. As aresult, it has been shown that a protein derived from the WT1 genehighly expressed in tumor cells is fragmented by intracellularprocessing and the resulting peptide forms a complex with an MHC class Imolecule which is displayed on the cell surface, and that cytotoxic Tcells (hereinafter also referred to as CTLs) recognizing such a complexcan be induced by WT1 peptide vaccination (Non-Patent Documents 7 to 9).It has also been shown that mice immunized with a WT1 peptide or a WT1cDNA reject implanted WT1 gene-expressing tumor cells in a high rate(Non-Patent Documents 7 and 10) but normal tissues endogenouslyexpressing the WT1 gene are not damaged by induced CTLs (Non-PatentDocument 7). Heretofore, it has been strongly suggested that it ispossible to induce WT1-specific CTLs in not only mice but also human,and that such CTLs have a cytotoxic activity against tumor cells highlyexpressing the WT1 gene, but have no cytotoxic activity against normalcells endogenously expressing the WT1 gene (Non-Patent Documents 7 and10 to 14).

On the other hand, it is reported that the presence of helper T cellsspecific to a cancer antigen is important in order to induce the CTLseffectively (Non-Patent Document 15). The helper T cells (CD4-positive Tcells) are induced, proliferated, and activated by recognizing a complexof an MHC class II molecule with an antigen peptide on antigenpresenting cells. Activated helper T cells produce cytokines such asIL-2, IL-4, IL-5, IL-6, or an interferon (IFN), and promoteproliferation, differentiation and maturation of B cells and othersubsets of T cells. Thus, it is considered that an antigen peptidebinding to an MHC class II molecule effectively activates CTLs andothers through induction of helper T cells and enhances an immunefunction (Non-Patent Document 16).

Heretofore, only an antigen peptide binding to HLA-DRB1*0401 andHLA-DRB1*0405 of an MHC class II molecule has been reported with respectto WT1 (Non-Patent Document 17 and Patent Document 1), and it wasnecessary to find antigen peptides to other subtypes.

PRIOR ART DOCUMENTS Non-Patent Documents

-   Patent Document 1: International Publication No. WO 2005/045027

Non-Patent Documents

-   Non-Patent Document 1: Daniel A. Haber et al., Cell. 1990 Jun. 29;    61(7): 1257-69.-   Non-Patent Document 2: Call K M et al., Cell. 1990 Feb. 9; 60(3):    509-20.-   Non-Patent Document 3: Menke A L et al., Int Rev Cytol. 1998; 181:    151-212. Review.-   Non-Patent Document 4: Yamagami T et al., Blood. 1996 Apr. 1; 87(7):    2878-84.-   Non-Patent Document 5: Inoue K et al., Blood. 1998 Apr. 15; 91(8):    2969-76.-   Non-Patent Document 6: Tsuboi A et al., Leuk Res. 1999 May; 23(5):    499-505.-   Non-Patent Document 7: Oka Y et al., J Immunol. 2000 Feb. 15;    164(4): 1873-80.-   Non-Patent Document 8: Melief C J et al., Immunol Rev. 1995 June;    145: 167-77.-   Non-Patent Document 9: Ritz J, J Clin Oncol. 1994 February; 12(2):    237-8.-   Non-Patent Document 10: Tsuboi A et al., J Clin Immunol. 2000 May;    20(3): 195-202.-   Non-Patent Document 11: Oka Y et al., Immunogenetics. 2000 February;    51(2): 99-107.-   Non-Patent Document 12: Ohminami H et al., Blood. 2000 Jan. 1;    95(1): 286-93.-   Non-Patent Document 13: Gao L et al., Blood. 2000 Apr. 1; 95(7):    2198-203.-   Non-Patent Document 14: Ohminami H et al., Blood. 2000 Jan. 1;    95(1): 286-93.-   Non-Patent Document 15: Cancer. Res. 62: 6438, 2002-   Non-Patent Document 16: J. Immunol. Immunother., 24: 195, 2001-   Non-Patent Document 17: Cancer. Immunol. Immunother. 51: 271, 2002

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention

Accordingly, an object to be achieved by the present invention is toprovide a peptide inducing WT1-specific helper T cells by binding tovarious MHC class II molecules, a polynucleotide encoding the peptide,WT1 helper T cells induced by the peptide, and a pharmaceuticalcomposition for treating/preventing cancer comprising them.

Means for Solving the Problems

The present inventors have intensively studied to achieve the aboveobject. As a result, they has found that a peptide having a portion of asequence of contiguous amino acids encoding a WT1 protein functions as acancer antigen helper peptide, in other words, the peptide is displayedon antigen presenting cells by binding to an MHC class II molecule andinduces WT1-specific helper T cells, and showed that the peptide can beused in a pharmaceutical composition for treating/preventing cancer.

Thus, the present invention provides:

(1) A peptide which has an amino acid sequence consisting of contiguousamino acids derived from a WT1 protein and induces WT1-specific helper Tcells by binding to an MHC class II molecule, wherein the amino acidsequence is selected from the group consisting of:

(a) the amino acid sequence depicted in SEQ ID NO:3;

(b) the amino acid sequence depicted in SEQ ID NO:4;

(c) the amino acid sequence depicted in SEQ ID NO:5; and

(d) an amino acid sequence in which one or several amino acids aresubstituted, deleted or added in the amino acid sequences depicted in(a) to (c);

(2) The peptide according to (1), wherein the amino acid sequence is theamino acid sequence depicted in SEQ ID NO:3;

(3) The peptide according to (1) or (2), wherein the MHC class IImolecule is selected from the group consisting of DRB1*0101, DRB1*0405,DRB1*0802, DRB1*0803, DRB1*0901, DRB1*1201, DRB1*1403, DRB1*1501,DRB1*1502, DPB1*0201, DPB1*0202, DPB1*0402, DPB1*0501, DPB1*0901,DQB1*0301, DQB1*0302, DQB1*0401, DQB1*0501, DQB1*0601, DQB1*0602, andDRB5*0102;

(4) The peptide according to (1) or (2), wherein the MHC class IImolecule is selected from the group consisting of DRB1*0101, DRB1*0405,DRB1*1502, DPB1*0201, DPB1*0202, and DQB1*0601;

(5) A polynucleotide encoding the peptide according to any one of (1) to(4);

(6) An expression vector comprising the polynucleotide according to (5);

(7) An antibody against the peptide according to any one of (1) to (4),or the polynucleotide according to (5);

(8) A pharmaceutical composition for treating or preventing cancer,comprising the peptide according to any one of (1) to (4), thepolynucleotide according to (5), or the vector according to (6);

(9) A method for treating or preventing cancer, which comprisesadministering an effective amount of the peptide according to any one of(1) to (4), the polynucleotide according to (5), or the vector accordingto (6) to a subject having the MHC class II molecule according to (3) or(4);

(10) Use of the peptide according to any one of (1) to (4), thepolynucleotide according to (5), or the vector according to (6) fortreating or preventing cancer;

(11) Antigen presenting cells which display the peptide according to anyone of (1) to (4) through the MHC class II molecule according to (3) or(4);

(12) A method for inducing antigen presenting cells, which includesculturing immature antigen presenting cells in the presence of thepeptide according to any one of (1) to (4), and inducing antigenpresenting cells, which display the peptide through the MHC class IImolecule according to (3) or (4), from the immature antigen presentingcells;

(13) WT1-Specific helper T cells which are induced by the peptideaccording to any one of (1) to (4);

(14) A method for inducing WT1-specific helper T cells, which comprisesculturing peripheral blood mononuclear cells in the presence of thepeptide according to any one of (1) to (4), and inducing WT1-specifichelper T cells from the peripheral blood mononuclear cells;

(15) A kit for inducing WT1-specific helper T cells, comprising, as anessential ingredient, the peptide according to any one of (1) to (4);

(16) A kit for preventing or treating cancer, comprising, as anessential ingredient, the peptide according to any one of (1) to (4),the polynucleotide according to (5), or the vector according to (6);

(17) A method for determining the presence or amount of WT1-specifichelper T cells in a subject having the MHC class II molecule accordingto (3) or (4), said method comprising the steps of:

(a) reacting the peptide according to any one of (1) to (4) with asample derived from the subject; and then

(b) determining the presence or amount of a cytokine contained in thesample.

Effects of the Invention

According to the present invention, it is possible to obtain WT1 helperpeptides which bind to many types of MHC class II molecules such asDRB1*0101, DRB1*0405, DRB1*0802, DRB1*0803, DRB1*0901, DRB1*1201,DRB1*1403, DRB1*1501, DRB1*1502, DPB1*0201, DPB1*0202, DPB1*0402,DPB1*0501, DPB1*0901, DQB1*0301, DQB1*0302, DQB1*0401, DQB1*0501,DQB1*0601, DQB1*0602, and DRB5*0102, a pharmaceutical composition fortreating/preventing cancer including them and the like. Thus, it becomespossible to induce WT1-specific helper T cells in vivo and in vitro invarious subjects (in particular, most Japanese have the abovemolecules). Since the WT1-specific helper T cells are induced by thepresent invention, it is also possible to activate T cells and B cellseffectively in cancer highly expressing the WT1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the results obtained by measuring cell proliferationafter stimulating each peptide-specific T cell line, which was preparedby pulsing with each of three peptides (mWT1₃₅, mWT1₈₆, and mWT1₂₉₄),with each peptide. In the drawing, the symbol “-” shows no peptidestimulation.

FIGS. 2A-2C show the results obtained by measuring cell proliferationafter stimulating each peptide-specific T cell line, which was preparedby pulsing with three peptides (mWT1₃₅ mWT1₈₆ and mWT1₂₉₄), with eachcorresponding peptide in the presence of an anti-MHC class I or IIantibody. In the drawing, the symbol “-” shows no peptide stimulation.The symbol “cpm” in the ordinate shows counts per minute.

FIG. 3 shows the results obtained by measuring cell proliferation ofeach WT1 peptide-specific T cell line in response to C1498 cells, C1498cells pulsed with three peptides (mWT1₃ ₅, mWT1₈ ₆, and mWT1₂ ₉ ₄), aswell as C1498 cells having forced expression of a WT1 protein. Thesymbol “cpm” in the ordinate shows counts per minute.

FIG. 4 shows the results obtained by measuring an IFN-γ producingability in each peptide-specific T cell line prepared by pulsing withthree peptides (mWT1₃ ₅, mWT1₈ ₆, and mWT1₂ ₉ ₄).

FIG. 5 shows the results obtained by measuring a CTL cytotoxic activityof three peptides (mWT1₃ ₅, mWT1₈ ₆, and mWT1₂ ₉ ₄).

shows the results of experiments carried out using RMAS cells pulsedwith a WT1₁ ₂ ₆ peptide (MHC class I-restricted peptide). ◯ shows theresults of experiments carried out using control RMAS cells.

FIG. 6 shows a time-series schematic drawing when carrying out tumorimplantation and immunization in a tumor implantation experiment.Immunization with an mWT1₃ ₅ helper peptide was carried out on the 7th,14th and 21st days after subcutaneous implantation of WT1-expressingleukemia cells to mice, and dissection was carried out on the 29th day.Downward white arrows show time points at which a control (PBS) wasintradermally administered (IFA/30 μl). Downward black arrows show timepoints at which an mWT1₃ ₅ helper peptide was intradermally administered(50 μM/IFA/30 μl).

FIG. 7 shows tumor sizes in mice immunized with an mWT1₃ ₅ helperpeptide and a proportion of disease-free mouse populations. In miceimmunized with an mWT1₃ ₅ helper peptide, 4 of 10 mice weredisease-free. On the other hand, in mice immunized with a control, therewas no disease-free mouse in 9 mice.

FIG. 8 shows a disease-free survival rate in mice immunized with anmWT1₃ ₅ helper peptide.

FIG. 9 shows cytotoxic activity of CTLs in mice immunized with an mWT1₃₅ helper peptide.

shows the results of experiments carried out using RMAS cells pulsedwith an mWT1₁ ₂ ₆ peptide (MHC I peptide). ◯ shows the results ofexperiments carried out using control RMAS cells. The numerical inparenthesis represents a tumor size (mm).

FIG. 10 shows cytotoxic activity of mWT1-specific CTLs in control mice.

shows the results of experiments carried out using RMAS cells pulsedwith an mWT1₁ ₂ ₆ peptide (MHC I peptide). ◯ shows the results ofexperiments carried out using control RMAS cells. The numeral inparenthesis represents a tumor size (mm).

FIG. 11 shows cytotoxic activity of mWT1₁ ₂ ₆ peptide-specific CTLs(left) and a proportion of WT1₁ ₂ ₆ tetramer-positive T cells (right)when an mWT1₃ ₅ peptide was administered.

FIG. 12 shows the results obtained by measuring cell proliferation byWT1₃ ₅ peptide stimulation in peripheral blood mononuclear cells of eachhealthy subject having MHC class II molecules.

FIG. 13 shows the results obtained by measuring cell proliferation whena Responder [PBMCs derived from a DRB1*0101/0405-, DPB1*0201/0402-, andDQB1*0401/0501-positive healthy subject (healthy subject A)] was treatedwith a Stimulator [PBMCs derived from a DRB1*0405/0901-,DPB1*0201/0501-, and DQB1*0303/0401-positive healthy subject (healthysubject B)]. The ordinate shows the amount of ³H-thymidine incorporated(cpm). The abscissa shows the types of various antibodies added (noantibody, anti-HLA-DR antibody, anti-HLA-DP antibody, and anti-HLA-DQantibody).

FIG. 14 shows the results obtained by measuring cell proliferation whena Responder [PBMCs derived from a DRB1*0101/0405-, DPB1*0201/0402-, andDQB1*0401/0501-positive healthy subject (healthy subject A)] was treatedwith a Stimulator [PBMCs derived from a DRB1*0405/0803-,DPB1*0202/0501-, and DQB1*0401/0601-positive healthy subject (healthysubject G)]. The ordinate shows the amount of ³H-thymidine incorporated(cpm). The abscissa shows the types of various antibodies added (noantibody, anti-HLA-DR antibody, anti-HLA-DP antibody, and anti-HLA-DQantibody).

FIG. 15 shows the results obtained by measuring cell proliferation whena Responder [PBMCs derived from a healthy subject having DRB10101/0405,DPB10201/0402, and DQB1*0401/0501 (healthy subject A)] was treated witha Stimulator [PBMCs derived from a DRB10101/0803-, DPB1*0501/-, andDQB1*0501/0601-positive healthy subject (healthy subject H)]. Theordinate shows the amount of ³H-thymidine incorporated (cpm). Theabscissa shows the types of various antibodies added (no antibody,anti-HLA-DR antibody, anti-HLA-DP antibody, and anti-HLA-DQ antibody).

FIG. 16 shows the results obtained by measuring an IFN-γ producingability when a Responder [PBMCs derived from a DRB10405/0803-,DPB1*0202/0501-, and DQB1*0401/0601-positive healthy subject (healthysubject G)] was treated with a Stimulator (L cells having a DQB1*0601gene introduced). The ordinate shows a proportion of an amount of IFN-γin T cells. The abscissa shows the presence or absence (+ or −) of apulse with a WT1₃ ₅ peptide.

FIG. 17 shows the results obtained by measuring cell proliferation whena Responder [PBMCs derived from a DRB1*1502/1502-, DPB1*0201/0901-, andDQB10601/0601-positive healthy subject (healthy subject D)] was treatedwith a Stimulator (PBMCs derived from the same healthy subject as in theResponder). The ordinate shows the amount of ³H-thymidine incorporated(cpm). The abscissa shows the types of various antibodies added (noantibody, anti-HLA-DR antibody, anti-HLA-DP antibody, and anti-HLA-DQantibody).

FIG. 18 shows the results obtained by measuring an IFN-γ producingability when a Responder [PBMCs derived from a DRB1*0101/1501-,DPB1*0201/0402-, and DQB1*0501/0602-positive healthy subject (healthysubject I)] was treated with a Stimulator (PBMCs derived from the samehealthy subject as in the Responder). The ordinate shows a proportion ofan amount of IFN-γ in T cells. The abscissa shows the presence orabsence (+ or −) of a pulse with a WT1₃ ₅ peptide.

MODE FOR CARRYING OUT THE INVENTION

In one aspect, the present invention relates to a peptide having anamino acid sequence consisting of amino acids derived from a mouse orhuman WT1 protein. The WT1 gene is highly expressed, for example, inhematopoietic organ tumors such as leukemia; myelodysplastic syndrome,multiple myeloma, and malignant lymphoma; solid cancers such as stomachcancer, bowel cancer, lung cancer, breast cancer, germ-cell cancer,liver cancer, skin cancer, bladder cancer, prostate cancer, uteruscancer, cervical cancer, and ovary cancer. Thus, the peptide of thepresent invention is present in cancer cells expressing the WT1 gene ina large amount.

The peptide of the present invention is a peptide which has an aminoacid sequence consisting of contiguous amino acids derived from thehuman WT1 protein depicted in SEQ ID NO:2, retains an ability to bind tothe MHC class II molecules as shown below, and has an ability to induceWT1-specific helper T cells. There is no particular limitation on theamino acid sequence and length of the peptide of the present inventionas long as the peptide has the above features. However, too long peptideis susceptible to a protease action, and too short peptide can not bindto a peptide accommodating groove well. The length of the peptide of thepresent invention is preferably 10 to 25 amino acids, more preferably 15to 21 amino acids, further preferably 16 to 20 amino acids, for example,of 17 amino acids, 18 amino acids, or 19 amino acids. Specific examplesof the peptide of the present invention are those having the amino acidsequence depicted in SEQ ID NO:3; the amino acid sequence depicted inSEQ ID NO:4; and the amino acid sequence depicted in SEQ ID NO:5.

Also, the peptide of the present invention includes variants of theabove peptides. The variants may contain, for example, a peptideselected from the group consisting of peptides having an amino acidsequence which has substitution, deletion or addition of several aminoacids, for example, 1 to 9, preferably 1 to 5, 1 to 4, 1 to 3, morepreferably 1 to 2 amino acids, further preferably one amino acid in oneof the above amino acid sequences. Substitution of amino acids inpeptides may be carried out at any positions and with any types of aminoacids. Conservative amino acid substitution is preferred. For example, aGlu residue may be substituted with an Asp residue, a Phe residue with aTyr residue, a Leu residue with an Ile residue, an Ala residue with aSer residue, and a His residue with an Arg residue. Addition or deletionof amino acids may be carried out preferably at the N-terminus and theC-terminus in peptides, but may be carried out in an interior sequence.A preferred specific example of the peptide of the present invention hasthe sequence of SEQ ID NO:3. In this regard, all the above peptides mustretain an ability to bind to an MHC class II molecule and have anability to induce WT1-specific helper T cells.

In this connection, the MHC class II molecule to which the peptide ofthe present invention binds may belong to any subclass of HLA-DR,HLA-DQ, and HLA-DP. Preferably, the MHC class II molecule is oneselected from the group consisting of DRB1*0101, DRB1*0405, DRB1*0802,DRB1*0803, DRB1*0901, DRB1*1201, DRB1*1403, DRB1*1501, DRB1*1502,DPB1*0201, DPB1*0202, DPB1*0402, DPB1*0501, DPB1*0901, DQB1*0301,DQB1*0302, DQB1*0401, DQB1*0501, DQB1*0601, DQB1*0602, and DRB5*0102.More preferably, the MHC class II molecule is DRB1*0101, DRB1*0405,DRB1*1403, DRB1*1502, DPB1*0201, DPB1*0202, DPB1*0901, DQB1*0301,DQB1*0601 or DRB5*0102, and most preferably, DRB10101, DRB1*0405,DRB1*1502, DPB1*0201, DPB1*0202, or DQB1*0601. In the presentspecification, a peptide which retains an ability to bind to an MHCclass II molecule and has an ability to induce WT1-specific helper Tcells is referred to as a WT1 helper peptide. Also, in the Examplesdescribed below, a peptide having the amino acid sequence depicted inSEQ ID NO:3 is referred to as a WT1₃ ₅ peptide, WT1₃ ₅ helper peptide orWT1₃ ₅ peptide.

Also, the peptide of the present invention may be a peptide having anamino acid sequence consisting of contiguous amino acids derived fromthe mouse WT1 protein depicted in SEQ ID NO:1, and the above amino acidsequence may be a peptide (SEQ ID NO:6) in which an amino acid residueat position 9 in the amino acid sequence depicted in SEQ ID NO:4 issubstituted with leucine; or a peptide (SEQ ID NO:7) in which an aminoacid residue at position 11 in the amino acid sequence depicted in SEQID NO:5 is substituted with serine. Moreover, the peptide of the presentinvention may contain a peptide selected from the group consisting ofpeptides having an amino acid sequence which has substitution, deletionor addition of several amino acids, for example, 1 to 9, preferably 1 to5, 1 to 4, 1 to 3, more preferably 1 to 2 amino acids, furtherpreferably one amino acid in the amino acid sequence depicted in SEQ IDNO:6 or SEQ ID NO:7. In the Examples described below, a peptide havingthe amino acid sequence depicted in SEQ ID NO:6 is also referred to asan mWT1₈ ₆ peptide or an mWT1₈ ₆ helper peptide, and a peptide havingthe amino acid sequence depicted in SEQ ID NO:7 as an mWT1₂ ₉ ₄ peptideor an mWT1₂ ₉ ₄ helper peptide.

The peptide of the present invention may be derived from a WT1 protein,and may consist of the above sequence of contiguous amino acids orcomprise the sequence. Thus, the peptide of the present invention maybe, for example, a peptide consisting of the above amino acid sequenceitself, or a WT1 protein comprising the above amino acid sequence or aportion thereof. Also, the peptide of the present invention may be thatobtained by modification of the above amino acid sequence. Amino acidresidues in the above amino acid sequence can be modified by a knownmethod. Such modification may be, for example, esterification,alkylation, halogenation, phosphorylation, sulfonation, amidation andthe like on a functional group in a side chain of an amino acid residueconstituting a peptide. Also, it is possible to bind various substancesto the N-terminus and/or C-terminus of a peptide containing the aboveamino acid sequence. For example, an amino acid, a peptide, an analogthereof and the like may be bound to the peptide. In case thesesubstances are bound to the peptide of the present invention, they maybe treated, for example, by an enzyme in vivo and the like or by aprocess such as intracellular processing so as to finally generate apeptide consisting of the above amino acid sequence, which is displayedon cell surface as a complex with an MHC class IT molecule, therebybeing able to obtain an induction effect of helper T cells. Thesesubstances may be those regulating solubility of the peptide of thepresent invention, those improving stability of the peptide such asprotease resistance, those allowing specific delivery of the peptide ofthe present invention, for example, to a given tissue or organ, or thosehaving an enhancing action of an uptake efficiency of antigen presentingcells or other action. Also, these substances may be those increasing anability to induce CTLs, for example, helper peptides other than thepeptide of the present invention.

The modification of the peptide of the present invention may bemodification of an amino group on an N-terminal amino acid or of acarboxyl group on a C-terminal amino acid of the peptide. Modifyinggroups of an amino group on an N-terminal amino acid include, forexample, one to three alkyl groups having 1 to 6 carbon atoms, phenylgroups, cycloalkyl groups, and acyl groups. Specific examples of theacyl group include an alkanoyl group having 1 to 6 carbon atoms, analkanoyl group having 1 to 6 carbon atoms substituted with a phenylgroup, a carbonyl group substituted with a cycloalkyl group having 5 to7 carbon atoms, an alkylsulfonyl group having 1 to 6 carbon atoms, aphenylsulfonyl group, an alkoxycarbonyl group having 2 to 6 carbonatoms, an alkoxycarbonyl group substituted with a phenyl group, acarbonyl group substituted with a cycloalkoxy group having 5 to 7 carbonatoms, a phenoxycarbonyl group and the like. Peptides havingmodification of a carboxyl group on a C-terminal amino acid include, forexample, esterified and amidated peptides. Specific examples of theester include an alkyl ester having 1 to 6 carbon atoms, an alkyl esterhaving 0 to 6 carbon atoms substituted with a phenyl group, a cycloalkylester having 5 to 7 carbon atoms and the like, and specific examples ofthe amide include an amide, an amide substituted with one or two alkylgroups having 1 to 6 carbon atoms, an amide substituted with one or twoalkyl groups having 0 to 6 carbon atoms substituted with a phenyl group,an amide forming a 5- to 7-membered azacycloalkane including a nitrogenatom of the amide group, and the like.

Also, the modification of the peptide of the present invention may becarried out by binding amino acid residues to each other through a bondother than a peptide bond such as a carbon-carbon bond, acarbon-nitrogen bond, and a carbon-sulfur bond. Moreover, the peptide ofthe present invention may contain one or more D-amino acids.

The above-mentioned peptides, variant peptides and modified peptidesaccording to the present invention are illustrative only, and thoseskilled in the art can easily assume, prepare, evaluate and use othervariations of the above peptides.

The peptide of the present invention can be synthesized using a methodroutinely used in the art or a modified method thereof. Such a synthesismethod is disclosed, for example, in Peptide Synthesis, Interscience,New York, 1966; The Proteins, Vol. 2, Academic Press Inc., New York,1976; Peptide Synthesis, Maruzen Co., Ltd., 1975; Basis and Experimentsof Peptide Synthesis, Maruzen Co., Ltd., 1985; Development of Medicines(continuation), Vol. 14, Peptide Synthesis, Hirokawa Shoten Co., 1991and the like. Also, the peptide of the present invention can be preparedusing a genetic engineering technique on the basis of information of anucleotide sequence encoding the peptide of the present invention. Sucha genetic engineering technique is well known to those skilled in theart. Such a technique can be conducted according to a method describedin literatures [Molecular Cloning, T. Maniatis et al., CSH Laboratory(1983); DNA Cloning, DM. Glover, IRL PRESS (1985)] as described above ora method described below, and other methods.

It is possible to determine whether the peptide of the present inventionor a candidate peptide thereof binds to the above MHC class II moleculeand induces helper T cells, by a known method such as, for example, amethod described in Cancer Immunol. Immunother. 51:271 (2002), or amethod described in the Examples of the present specification, and othermethods.

Since the peptide of the present invention activates helper T cells(CD4-positive T cells), the peptide induces and maintainsdifferentiation of CTLs and exerts an action of activating effectorcells such as macrophages. Accordingly, it is possible to use thepeptide of the present invention for effective treatment or preventionof cancer.

In another aspect, the present invention relates to a polynucleotideencoding the above WT1 helper peptide (hereinafter also referred to as aWT1 polynucleotide). The polynucleotide of the present invention may bea DNA or an RNA. The base sequence of the polynucleotide of the presentinvention can be determined on the basis of the amino acid sequence ofthe above WT1 helper peptide. The polynucleotide can be prepared, forexample, by a method for DNA or RNA synthesis, a PCR method and thelike.

The polynucleotide of the present invention includes a polynucleotidewhich hybridizes with a complementary sequence of a polynucleotideencoding the peptide of the present invention under a stringentcondition and encodes a peptide having an activity comparable to that ofthe peptide of the present invention. As to the term “hybridize under astringent condition”, hybridization used herein can be carried outaccording to a conventional method described, for example, in MolecularCloning, 2nd edition, Sambrook J., Frisch E. F., Maniatis T., ColdSpring Harbor Laboratory press and the like. Also, the “stringentcondition” includes, for example, a condition wherein a hybrid is formedin a solution containing 6×SSC (10×SSC is a solution containing 1.5 MNaCl and 0.15 M trisodium citrate) and 50% formamide at 45° C. and thenwashed with 2×SSC at 50° C. (Molecular Biology, John Wiley & Sons, N.Y.(1989), 6.3.1-6.3.6) and the like.

In still another aspect, the present invention relates to an expressionvector comprising the above polynucleotide (hereinafter also referred toas a WT1 expression vector). The type of expression vectors, othersequences contained in addition to the above polynucleotide sequence andthe like can be appropriately selected depending on the type of hostsinto which the expression vectors are introduced, the purpose of theintroduction and the like. Examples of the expression vector includeplasmids, phage vectors, virus vectors and the like. In case the host isEscherichia coli cells, examples of the vector include plasmid vectorssuch as pUC118, pUC119, pBR322, and pCR3, as well as phage vectors suchas λZAPII, and Δgt11. In case the host is yeast cells, examples of thevector include pYES2, pYEUra3 and the like. In case the host is insectcells, examples of the vector pAcSGHisNT-A and the like. In case thehost is animal cells, examples of the vector include plasmid vectorssuch as pKCR, pCDM8, pGL2, pcDNA3.1, pRc/RSV, and pRc/CMV, virus vectorssuch as a retrovirus vector, an adenovirus vector, and anadeno-associated virus vector. The vector may optionally contain factorssuch as an expression-inducible promoter, a gene encoding a signalsequence, a marker gene for selection, and a terminator. Also, asequence expressed as a fusion protein with thioredoxin, a His tag, GST(glutathione S-transferase) and the like may be added to the vector foreasy isolation and purification. In this case, it is possible to use aGST-fused protein vector (pGEX4T, etc.) having a suitable promoter (lac,tac, trc, trp, CMV, SV40 early promoter, etc.) functional in host cells,a vector (pcDNA3.1/Myc-His, etc.) having a tag sequence such as Myc andHis, and also a vector (pET32a) expressing a fusion protein withthioredoxin and a His tag and the like.

When the expression vector of the present invention is administered to asubject to produce a WT1 helper peptide in vivo, WT1-specific helper Tcells induced by the peptide produce various cytokines (for example,IL-2, IL-4, IL-5, IL-6, or an interferon (IFN), etc.), and promoteproliferation, differentiation and maturation of B cells and other Tcells. Accordingly, tumor cells which have an MHC class I molecule andhighly express WT1 can be damaged specifically using the WT1 expressionvector of the present invention.

In another aspect, the present invention relates to an antibody againstthe above WT1 helper peptide or a polynucleotide encoding the peptide(hereinafter also referred to as a WT1 antibody). The antibody of thepresent invention may be either of a polyclonal antibody or a monoclonalantibody. A method for preparing such an antibody is already known, andthe antibody of the present invention can be prepared according to sucha conventional method as well (Current protocols in Molecular Biology,Ausubel et al. (ed.), 1987, John Wiley and Sons (pub.), Section11.12-11.13, Antibodies; A Laboratory Manual, Lane, H. D. et al. (ed.),Cold Spring Harber Laboratory Press (pub.), New York, 1989).

The present invention relates to a pharmaceutical composition fortreating or preventing cancer, comprising the above WT1 helper peptide,WT1 polynucleotide, or WT1 expression vector. The WT1 gene is highlyexpressed, for example, in hematopoietic organ tumors such as leukemia,myelodysplastic syndrome, multiple myeloma, and malignant lymphoma, aswell as in solid cancers such as stomach cancer, bowel cancer, lungcancer, breast cancer, germ-cell cancer, liver cancer, skin cancer,bladder cancer, prostate cancer, uterus cancer, cervical cancer, andovary cancer, and therefore, it is possible to use the pharmaceuticalcomposition of the present invention for treating or preventing cancerexpressing the WT1 gene. When the pharmaceutical composition of thepresent invention is administered to a subject having an MHC class IImolecule, WT1-specific helper T cells induced by a WT1 helper peptidecontained in the pharmaceutical composition produce various cytokines(for example, IL-2, IL-4, IL-5, IL-6, or an interferon (IFN), etc.), andpromote proliferation, differentiation and maturation of B cells andother subsets of T cells. Accordingly, tumor cells which have an MHCclass I molecule and highly express WT1 can be damaged specificallyusing the peptide of the present invention.

The pharmaceutical composition of the present invention may comprise,for example, a carrier, an excipient and the like, in addition to theabove WT1 helper peptide, WT1 polynucleotide, or WT1 expression vectoras an effective component. The WT1 helper peptide contained in thepharmaceutical composition of the present invention induces WT1-specifichelper T cells, and thus the pharmaceutical composition of the presentinvention may comprise a suitable adjuvant or may be administeredtogether with a suitable adjuvant in order to enhance the inductionefficiency. Examples of preferred adjuvant include, but are not limitedto, a Freund's complete or incomplete adjuvant, aluminium hydroxide andthe like. Also, the pharmaceutical composition of the present inventionmay also comprise a known cancer antigen peptide other than the aboveWT1 helper peptide such as, for example, a WT1₁ ₂ ₆ peptide inducingWT1-specific CTLs, as an effective component (Oka et al, “Cancerimmunotherapy targeting Wilms' tumor gene WT1 product”, Journal ofImmunology, 164:1873-1880, 2000; and Oka et al., “Human cytotoxicT-lymphocyte responses specific for peptides of the wild-type Wilms'tumor gene (WT1) product”, Immunogenetics, 51: 99-107, 2000).

Moreover, the pharmaceutical composition of the present invention may beadministered in combination with a known cancer antigen peptide. Forexample, a known cancer antigen peptide, for example, a WT1₁ ₂ ₆ peptidecan be administered before or after the administration of thepharmaceutical composition of the present invention. The pharmaceuticalcomposition of the present invention has a feature that activates Bcells or other T cells by inducing WT1-specific helper T cells, andtherefore, it is possible to further enhance an activity of CTLs inducedby administering a known cancer antigen peptide, and to remarkablyincrease therapeutic effects.

A method for administering the pharmaceutical composition of the presentinvention can be appropriately selected depending on conditions such asthe type of diseases, the state of subjects, and the targeted sites.Examples of the administration method includes, but are not limited to,intradermal administration, subcutaneous administration, intramuscularadministration, intravenous administration, transnasal administration,oral administration and the like. Also, the administration method may bea lymphocyte therapy or a DC (dendritic cell) therapy. The amount of apeptide contained in the pharmaceutical composition of the presentinvention, the form and administration frequency of the pharmaceuticalcomposition and the like can be appropriately selected depending onconditions such as the type of diseases, the state of subjects, and thetargeted sites. In general, the amount of a peptide administered perdose is 0.0001 mg to 1000 mg, and preferably 0.001 mg to 10,000 mg.

In another aspect, the present invention relates to a method fortreating or preventing cancer, which comprises administering aneffective amount of the above pharmaceutical composition to a subjecthaving the above MHC class II molecule. Cancers to be treated orprevented may be any cancers as long as they express the WT1 gene andinclude, for example, hematopoietic organ tumors such as leukemia,myelodysplastic syndrome, multiple myeloma, and malignant lymphoma, aswell as solid cancers such as stomach cancer, bowel cancer, lung cancer,breast cancer, germ-cell cancer, liver cancer, skin cancer, bladdercancer, prostate cancer, uterus cancer, cervical cancer, and ovarycancer.

In another aspect, the present invention relates to use of the above WT1helper peptide, WT1 polynucleotide, or WT1 expression vector fortreating or preventing cancer.

In still another aspect, the present invention relates to use of the WT1helper peptide for preparing a pharmaceutical composition for treatingor preventing cancer.

In still another aspect, the present invention relates to use of the WT1polynucleotide or WT1 expression vector for preparing a pharmaceuticalcomposition containing the above WT1 polynucleotide or WT1 expressionvector.

In another aspect, the present invention relates to cells including theabove WT1 helper peptide, WT1 polynucleotide, or WT1 expression vector.The cells of the present invention can be prepared, for example, bytransforming host cells such as Escherichia coli cells, yeast cells,insect cells, and animal cells using the above expression vector.Transformation of host cells with an expression vector can be carriedout using various methods properly selected. The peptide of the presentinvention can be prepared by culturing transformed cells, and recoveringand purifying a WT1 helper peptide produced.

In still another aspect, the present invention relates to antigenpresenting cells (for example, dendritic cells, B-lymphocytes,macrophages, etc.), which display the above WT1 helper peptide throughthe above MHC class II molecule. The antigen presenting cells of thepresent invention are induced by the above WT1 helper peptide.WT1-specific helper T cells are efficiently induced using the antigenpresenting cells of the present invention.

In still another aspect, the present invention relates to a method forinducing antigen presenting cells which display a WT1 helper peptidethrough an MHC class II molecule, said method comprising culturingimmature antigen presenting cells in the presence of a WT1 helperpeptide, and inducing antigen presenting cells, which display the WT1helper peptide through the above MHC class II molecule, from theimmature antigen presenting cells. In the present specification, theimmature antigen presenting cells refer to cells which can becomeantigen presenting cells such as, for example, dendritic cells,B-lymphocytes, and macrophages upon maturation. Subjects from which theimmature antigen presenting cells derive may be any subjects as long asthey have the above MHC class II molecule. Since the immature antigenpresenting cells are contained, for example, in peripheral bloodmononuclear cells and the like, such cells may be cultured in thepresence of the above WT1 helper peptide.

In another aspect, the present invention relates to a method fortreating or preventing cancer, which comprises administering antigenpresenting cells, which display a WT1 helper peptide through the aboveMHC class II molecule, to a subject having the same molecule as theabove MHC class II molecule. The administration method of the antigenpresenting cells can be appropriately selected depending on conditionssuch as the type of diseases, the state of subjects, and the targetedsites. Examples of the method include, but are not limited to,intravenous administration, intradermal administration, subcutaneousadministration, intramuscular administration, transnasal administration,oral administration and the like.

In still another aspect, the present invention relates to a method forpreventing or treating cancer by induction of antigen presenting cellswhich display a WT1 helper peptide through the above MHC class IImolecule, said method comprising the steps of:

(a) reacting a sample with a nucleotide sequence encoding an amino acidsequence (SEQ ID NO:2) of a WT1 protein or a nucleic acid having apartial sequence thereof or the above WT1 helper peptide;

(b) obtaining antigen presenting cells which display a WT1 helperpeptide contained in the sample through the above MHC class II molecule;and

(c) administering the antigen presenting cells to a subject having thesame molecule as the above MHC class II molecule.

Samples in the above method may be any samples as long as they have apossibility of containing lymphocytes or dendritic cells and include,for example, subject-derived samples such as blood, cell culturesolutions and the like. The reaction in the above method may be carriedout using a conventional technique, and preferably usingelectroporation. Obtainment of the antigen presenting cells can becarried out using a method known to those skilled in the art. Culturingconditions of cells in a sample in each step can be determined properlyby those skilled in the art. The administration method of the antigenpresenting cells may be as described above.

In further aspect, the present invention relates to WT1-specific helperT cells induced by the above WT1 helper peptide. The helper T cells ofthe present invention are induced, proliferated, and activated whenrecognizing a complex of a WT1 helper peptide with an MHC class IImolecule. The activated WT1-specific helper T cells produce cytokinessuch as IL-2, IL-4, IL-5, IL-6, or an interferon (IFN), and promoteproliferation, differentiation and maturation of B cells and othersubsets of T cells. Accordingly, tumor cells which have an MHC class Imolecule and highly express WT1 can be damaged specifically using thehelper T cells of the present invention.

In another aspect, the present invention relates to a method forinducing WT1-specific helper T cells, which comprises culturingperipheral blood mononuclear cells in the presence of a WT1 helperpeptide, and inducing the WT1-specific helper T cells from theperipheral blood mononuclear cells. Subjects from which the peripheralblood mononuclear cells derive may be any subjects as long as they havethe above MHC class II molecule. By culturing the peripheral bloodmononuclear cells in the presence of a WT1 helper peptide, WT1-specifichelper T cells are induced from precursor cells of helper T cells in theperipheral blood mononuclear cells. It is possible to treat or preventhematopoietic organ tumors and solid cancers in a subject byadministering the WT1-specific helper T cells obtained by the presentinvention to a subject having the above MHC class II molecule. In thisconnection, the peripheral blood mononuclear cells in the presentspecification include immature antigen presenting cells which areprecursor cells of antigen presenting cells (for example, precursorcells of dendritic cells, B-lymphocytes, macrophages, etc.) Since theimmature antigen presenting cells are contained, for example, inperipheral blood mononuclear cells and the like, such cells may becultured in the presence of the above WT1 helper peptide.

In still another aspect, the present invention relates to a kit forinducing WT1-specific helper T cells, comprising the above WT1 helperpeptide as an essential ingredient. Preferably, the kit is used in theabove method for inducing WT1-specific helper T cells. The kit of thepresent invention may comprise, for example, an obtaining means ofperipheral blood mononuclear cells, an adjuvant, a reaction vessel andothers, in addition to the above WT1 helper peptide. In general, the kitis accompanied with an instruction manual. It is possible to induceWT1-specific helper T cells efficiently using the kit of the presentinvention.

In still another aspect, the present invention relates to a method fortreating or preventing cancer, which comprises administeringWT1-specific helper T cells to a subject having the above MHC class IImolecule. The administration method of the WT1-specific helper T cellscan be appropriately selected depending on conditions such as the typeof diseases, the state of subjects, and the targeted sites. Examples ofthe administration method includes, but are not limited to, intravenousadministration, intradermal administration, subcutaneous administration,intramuscular administration, transnasal administration, oraladministration and the like.

Furthermore, the present invention relates to a kit for preventing ortreating cancer, comprising the above WT1 helper peptide, WT1polynucleotide, or WT1 expression vector as an essential ingredient. Thekit is a kit characterized by induction of antigen presenting cellswhich display the above WT1 helper peptide through the above MHC classII molecule. Also, the kit of the present invention may comprise, forexample, an obtaining means of samples, a reaction vessel and others, inaddition to the above essential ingredient. In general, the kit isaccompanied with an instruction manual. Antigen presenting cells whichdisplay a WT1 helper peptide through the above MHC class II molecule canbe obtained efficiently using the kit of the present invention, and usedfor treating or preventing cancer by their administration.

In another aspect, the present invention relates to a method fordetermining the presence or amount of WT1-specific helper T cells in asubject having the above MHC class II molecule, said method comprisingthe steps of:

(a) reacting a complex of the above WT1 helper peptide with the aboveMHC class II molecule with a sample derived from the subject; and then

(b) determining the presence or amount of helper T cells recognizing thecomplex contained in the sample.

Samples derived from subjects may be any samples as long as they have apossibility of containing lymphocytes and include, for example, bodyfluids such as blood and lymph fluid, tissues and the like. The complexof WT1 helper T cells with an MHC class II molecule may be, for example,in the form of tetramer, pentamer and the like, for example, using amethod known to those skilled in the art such as a biotin-streptavidinmethod. The presence or amount of helper T cells recognizing such acomplex can be determined by a method known to those skilled in the art.In this aspect of the present invention, the above complex may belabeled. As a label, known labels such as a fluorescent label and aradioactive label can be used. By labeling, the presence or amount ofhelper T cells can be determined simply and rapidly. Using a method ofthis aspect of the present invention, it becomes possible to make adiagnosis, a prognosis and the like of cancer.

Accordingly, the present invention also provides a compositioncomprising a complex of a WT1 helper peptide with the above MHC class IImolecule for determining the presence or amount of WT1-specific helper Tcells in a subject having the above MHC class II molecule.

Also, the present invention provides a kit comprising a complex of a WT1helper peptide with the above MHC class II molecule for determining thepresence or amount of WT1-specific helper T cells in a subject havingthe above MHC class II molecule.

In still another aspect, the present invention relates to a method fordetermining the presence or amount of WT1-specific helper T cells in asubject having the above MHC class II molecule, said method comprisingthe steps of:

(a) reacting the above WT1 helper peptide with a sample derived from thesubject; and then

(b) determining the presence or amount of a cytokine contained in thesample.

Samples derived from subjects may be any samples as long as they have apossibility of containing lymphocytes and include, for example,peripheral blood mononuclear cells, blood, body fluids, tissues andothers, and preferably peripheral blood mononuclear cells. The reactionin the above step (a) can be carried out by reacting the above WT1helper peptide in the above sample derived from a subject using aconventional technique. Culturing conditions of cells in a sample ineach step can be determined properly by those skilled in the art. Thepresence or amount of a cytokine contained in a sample can be measuredby a method known to those skilled in the art. The cytokine may be onecapable of being induced by helper T cells such as interferon-γ andinterleukin-10. In this aspect of the present invention, the abovecytokine may be labeled. As a label, known labels such as a fluorescentlabel and a radioactive label can be used. Using the presence or amountof the above cytokine as an indicator, it becomes possible to determinethe presence or amount of WT1-specific helper T cells simply andrapidly.

In further aspect, the present invention relates to a method forobtaining WT1-specific helper T cells using a complex of a WT1 helperpeptide with the above MHC class II molecule, said method comprising thesteps of:

(a) reacting a sample with the complex; and

(b) obtaining helper T cells which are contained in the sample andrecognize the complex.

The complex of a WT1 helper peptide with the above MHC class II moleculeis as described above. Samples may be any samples as long as they have apossibility of containing lymphocytes and include, for example,subject-derived samples such as blood, cell culture solutions and thelike. Obtainment of helper T cells recognizing the complex can becarried out, for example, using a method known to those skilled in theart such as FACS and MACS. It is possible to culture the resultingWT1-specific helper T cells and to use them for treating or preventingvarious cancers.

Accordingly, the present invention also relates to WT1-specific helper Tcells, which can be obtained by a method for obtaining WT1-specifichelper T cells using a complex of a WT1 helper peptide with the aboveMHC class II molecule.

Moreover, the present invention relates to a kit for obtainingWT1-specific helper T cells, comprising a complex of a WT1 helperpeptide with the above MHC class II molecule.

In still another aspect, the present invention relates to a method fordiagnosing cancer, which comprises using the above WT1-specific helper Tcells, the above antigen presenting cells which display a WT1 helperpeptide through the above MHC class II molecule, or the above WT1antibody. Preferably, the WT1-specific helper T cells are used for themethod for diagnosing cancer of the present invention. For example, theabove helper T cells, antigen presenting cells or antibody can beincubated with a sample derived from a subject having the above MHCclass II molecule, or administered to a subject having the above MHCclass II molecule, and then, for example, the location, site, amount andthe like of the helper T cells, antigen presenting cells or antibody canbe determined to diagnose cancer. The above helper T cells, antigenpresenting cells or antibody may be labeled. By labeling, it is possibleto carry out the method for diagnosing cancer of the present inventionefficiently.

In still another aspect, the present invention relates to a kit fordiagnosing cancer, comprising the above WT1-specific helper T cells,antigen presenting cells which display a WT1 helper peptide through theabove MHC class II molecule, or an antibody against a WT1 helper peptideor an antibody against a polynucleotide encoding the peptide, as anessential ingredient.

The present invention will be described specifically and described indetail below by way examples, but they should not be construed aslimiting the present invention.

Example 1

Selection of Candidate WT1 Peptides Binding to MHC Class II Molecules

In order to search peptide sequences which bind to MHC class IImolecules, a method as shown by Rammensee et al. was used (Rammensee etal, Immunogenetics 41:178-228, 1995). Specifically, selection wascarried out using the programs described in the right end column in theTables together with the law of Rammensee et al. By the method, WT1₃ ₅peptides were narrowed down to peptide sequences as shown in Tables 1and 2, WT1₆ peptides to peptide sequences as shown in Tables 3 and 4,and WT1₂ ₉ ₄ peptides to peptide sequences as shown in Tables 5 and 6.The left end column in Tables 1 to 6 shows “suitability” as a candidatepeptide sequence. The more the number of “◯” is, the higher thesuitability is in the law of Rammensee et al. No mark shows poorsuitability. Also, the group of amino acids in parenthesis of the columnof “candidate peptide sequences binding to MHC class II molecules” inTables 1 to 6 shows that one amino acid can be selected from the groupof amino acids listed in the parenthesis. For example, the description[FLM] means one amino acid selected from the group of amino acids F, Land M. Also, the description [VYI(AL)] means one amino acid selectedfrom the group of amino acids V, Y and I, or one amino acid selectedfrom the group of amino acids A and L. “x” shows that it may be anyamino acid. The right end column shows “program name” of programs usedfor listing candidate peptide sequences.

TABLE 1Candidate peptide sequences binding to various MHC class II molecules (WT1₃₅ peptides)Types of MHC class  Candidate peptide sequences binding Program  SEQ IDSuitability II molecules to MHC class II molecules name NO: ○○○DPA1*0102/DPB1*0201 [FLMVWY]xxx[FLMY]xx[IAMV] SYFPEITHI  9DPA1*0103/DPB1*0201 [YLVFK]xx[DSQT]x[YFWV]xx[LVI] Marsh2000,  10Chicz 1997 ○ DPA1*0103/DPB1*0201 [FLM]xxx[FL]xx[IA] Marsh2000,  11Rotzschke  1994 ○ DPA1*0201/DPB1*0401 [FLYM(IVA)]xxxxx[FLY(MVIA)]xxMarsh2000 12 [VYI(AL)] ○ DPA1*0201/DPB1*0401[FLYMIVA]xxxxx[FLYMVIA]xx[VYIAL] SYFPEITHI 13 DPA1*0201/DPB1*0901[RK]xxxx[AGL]xx[LV] Marsh2000 14 DPB1*0301 x[R]xxxxxxx Marsh2000 15DQA1:0101/DQB1*0501 [L]xxx[YFW] Marsh2000 16 ○ DQA1:0102/DQB1*0602xxxxx[LIV(APST)]xx[AGST(LIVP)] Marsh2000 17 ○ DQA1:0301/DQB1*0301xx[AGST]x[AVLI] Marsh2000 18 DQA1:0301/DQB1*0301 [DEW]xx[AGST]x[ACLM]SYFPEITHI 19 DQA1:0301/DQB1*0302 [RK]xxx[AG]xx[NED] Marsh2000 20 ○DQA1:0301/DQB1*0302 [TSW]xxxxxxx[RE] SYFPEITHI 21 ○○○DQA1:0501/DQB1*0201 [FWYILV]xx[DELVIH]x[PDE(H)][ED]x Marsh2000 22[FYWVILM] ○○○ DQA1:0501/DQB1*0201 [FWYILV]xx[DELVIH]x[PDEHPA][DE]xSYFPEITHI 23 [FYWVILM] ○○○ DQA1:0501/DQB1*0301[FYIMLV]xxx[VLIMY]x[YFMLVI] Marsh2000 24 ○ DQA1:0501/DQB1*0301[WYAVM]xx[A]x[AIVTS]xxx[QN] SYFPEITHI 25 ○○○ DQB1*0602[AFCILMNQSTVWYDE]x[AFGILMNQSTWYCDE]  SYFPEITHI 26[AFGILMNQSTWY]x[LIVAPST]xx[ASTGLIVP] ○○○ DRB1*0101[YFWLIMVA]xx[LMAIVN]x[AGSTCP]xx Marsh2000 27 [LAIVNFYMW] ○○○ DRB1*0101[YVILFIAMW]xx[LAIVMNQ]x[AGSTCP]xx SYFPEITHI 28 [LAIVNFY] DRB1*0102[ILVM]xx[ALM]x[AGSTCP]xx[ILAMYW] Marsh2000 29 DRB1*0102[ILVM]xx[ALM]x[AGSTP]xx[ILAMYW] SYFPEITHI 30 DRB1*0301[LIFMV]xx[D]x[KR(EQN)]x[L][YLF] Marsh2000,  31 Malcherek  1993 DRB1*0301[LIFMV]xx[D]x[KREQN]xx[YLF] SYFPEITHI 32 DRB1*0301 or DRB3*0201[FILVY]xx[DNQT] Marsh2000,  33 Chicz 1992 DRB1*0401 [FLV]xxxxxxx[NQST]Marsh2000 34 ○○○ DRB1*0401 or DRB4 [FYWILVM]xx[FWILVADE]x[NSTQHR]xx[K]Marsh2000,  35 Friede 1996 ○ DRB1*0401 or DRB4*0101 [FYW]xxxxxxx[ST]Marsh2000,  36 Verreck  1995 ○○○ DRB1*0401 or DRB4*0101[FYWILVM]xx[PWILVADE]x[NSTQHR] SYFPEITHI 37DEHKNQRSTYACILMV]x[DEHKNQRSTYACILMV] DRB1*0402 or DRB4[VILM]xx[YFWILMRNH]x[NSTQHK]x Marsh2000 38 [RKHNQP]x[H]DRB1*0402 or DRB4 [VILM]xx[YFWILMRN]x[NQSTK][RKHNQP]x SYFPEITHI 39[DEHLNQRSTYCILMVHA] ○○ DRB1*0404 or DRB4[VILM]xx[FYWILVMADE]x[NTSQR]xx[K] Marsh2000 40 ○○ DRB1*0404 or DRB4[VILM]xx[FYWILVMADE]x[NTSQR]xx[K] SYFPEITHI 40 ○○○ DRB1*0405 or DRB4[FYWVILM]xx[VILMDE]x[NSTQKD]xxx[DEQ] Marsh2000 41 ○○○ DRB1*0405 or DRB4[FYWVILM]xx[VILMDE]x[NSTQKD]xxx[DEQ] SYFPEITHI 41 DRB1*0405 or DRB4*0101[Y]xxxx[VT]xxx[D] Marsh2000 42 ○○○ DRB1*0407 or DRB4[FYW]xx[AVTK]x[NTDS]xxx[QN] Marsh2000 43 ○○○ DRB1*0407 or DRB4[FYW]xx[AVTK]x[NTDS]xxx[QN] SYFPEITHI 43

TABLE 2Candidate peptide sequences binding to various MHC class II molecules (WT1₃₅ peptides)Types of MHC  SEQ  class II Candidate peptide sequences binding Program ID Suitability molecules to MHC class II molecules name NO: DRB1*0701[FILVY]xxxx[NST] Marsh2000 44 ○ DRB1*0701[FYWILV]xx[DEHKNQRSTY]x[NST]x[VILYF] SYFPEITHI 45 DRB1*0801[FILTY]xxx[HKR] Marsh2000 46 ○ DRB1*0901 or  [YFWL]xx[AS] Marsh2000 47DRB4*0101 ○○○ DRB1*0901 or  [WYFL]xx[AVS] SYFPEITHI 48 DRB4*0101 ○○○DRB1*1101 [YF]xx[LVMAFY]x[RKHlxx[AGSP] Marsh2000 49 ○○○ DRB1*1101[WYF]xx[LVMAF]x[RKH]xx[AGSP] SYFPEITHI 50 DRB1*1101 or [YF]xxxx[RK]x[RK] Marsh2000 51 DRB3*0202 ○○○ DRB1*1104[ILV]xx[LVMAFY]x[RKH]xx[AGSP] Marsh2000 52 ○○○ DRB1*1104[ILV]xx[LVMAFY]x[RKH]xx[AGSP] SYFPEITHI 52 DRB1*1201 or [ILFY(V)]x[LNM(VA)]xx[VY(FIN)]xx[YFM(IV)] Marsh2000 53 DRB3DRB1*1201 or  [ILFYV]x[LMNVA]xx[VYFINA]xx[YFMIV] SYFPEITHI 54 DRB3 ○DRB1*1301 [IVF]xx[YWLVAM]x[RK]xx[YFAST] Marsh2000 55 ○ DRB1*1301[ILV]xx[LVMAWY]x[RK]xx[YFAST] SYETEITHI 56 DRB1*1301 or [ILV]xxxx[RK]xx[Y] Marsh2000 57 DRB3*0101 ○ DRB1*1302[YFVAI]xx[YWLVAM]x[RK]xx[YFAST] Marsh2000 58 ○ DRB1*1302[YFVAI]xx[LVMAWY]x[RK]xx[YFAST] SYFPEITHI 59 DRB1*1302 or [ILFY]xxxx[RF]xx[Y] Marsh2000 60 DRB3*1301 ○ DRB1*1501[LVI]xx[FYI]xx[ILVMF] Marsh2000 61 ○ DRB1*1501 [LVI]xx[FYI]xx[ILVMF]SYFPEITHI 61 DRB1*1501 or  [ILV]xxxxxxxx[HKR] Marsh2000 62 DRB5*0101 ○DRB3*0202 [YFIL]xx[N]x[ASPDE]xx[LVISG] Marsh2000 63 ○ DRB3*0202[YFIL]xx[N]x[ASPDE]xx[LVISG] SYFPEITHI 63 ○ DRB3*0301[ILV]xx[N]x[ASPDE]xx[ILV] Marsh2000 64 ○ DRB3*0301[ILV]xx[N]x[ASPDE]xx[ILV] SYFPEITHI 64 ○○ DRB5*0101[FYLM]xx[QVIM]xxxx[RK] Marsh2000 65 ○○ DRB5*0101 [FYLM]xx[QVIM]xxxx[RK]SYFPEITHI 65

TABLE 3Candidate peptide sequences binding to various MHC class II molecules (WT1₁₈₆ peptides)Types of MHC  SEQ  class II Candidate peptide sequences binding ProgramID Suitability molecules Serotype to MHC class II molecules name NO:DPA1*0102/ DPw2 unknown Marsh2000 DPB1*0201 DPA1*0102/ DPw2[FLMVWY]-x-x-x-[FLMY]-x-x-[IAMV] SYFPEITHI  9 DPB1*0201 DPA1*0103/ DPw2[FLM]-x-x-x-[FL]-x-x-[IA] Marsh2000 11 DPB1*0201 ○ DPA1*0103/ DPw2[YLVFK]-x-x-[DSQT]-x-[YFWV]-x-x-[LVI] Marsh2000 10 DPB1*0201 DPA1*0103/DPw2 unknown SYFPEITHI DPB1*0201 DPA1*0103/ DPw2 unknown SYFPEITHIDPB1*0201 ○○ DPA1*0201/ DPw4 [FLYM(IVA)]-x-x-x-x-x-[FLY(MVIA)]-x-x-Marsh2000 12 DPB1*0401 [VYI(AL)] ○○ DPA1*0201/ DPw4[FLYMIVA]-x-x-x-x-x-[FLYMVIA]-x-x-[VYIAL] SYFPEITHI 13 DPB1*0401 ○○○DPA1*0201/ [RK]-x-x-x-x-[AGL]-x-x-[LV] Marsh2000 14 DPB1*0901 DPB1*0301DPw3 x-[R]-x-x-x-x-x-x-x Marsh2000 15 DPB1*0301 DPw3 unknown SYFPEITHIDQA1*0101/ DQ5(1) [L]-x-x-x-[YFW] Marsh2000 16 DQB1*0501 DQA1*0101/DQ5(1) unknown SYFPEITHI DQB1*0501 ○○○ DQA1*0102/ DQ6(1)x-x-x-x-x-[LIV(APST)]-x-x-[AGST(LIVP)] Marsh2000 17 DQB1*0602 DQA1*0301/DQ7(3) x-x-[AGST]-x-[AVLI] Marsh2000 18 DQB1*0301 ○○○ DQA1*0301/ DQ7(3)[DEW]-x-x-[AGST]-x-[ACLM] SYFPEITHI 19 DQB1*0301 ○ DQA1*0301/ DQ8(3)[RK]-x-x-x-x-[AG]-x-x-[NED] Marsh2000 66 DQB1*0302 DQA1*0301/ DQ8(3)[TSW]-x-x-x-x-x-x-x-[RE] SYFPEITHI 21 DQB1*0201 ○○○ DQA1*0501/ DQ2[FWYILV]-x-x-[DELVIH]-x-[PDE(H)]-[ED]-x- Marsh2000 22 DQB1*0201[FYWVILM] ○○○ DQA1*0501/ DQ2 [FWYILV]-x-x-[DELVIH]-x-[PDEHPA]-[DE]-x-SYFPEITHI 67 DQB1*0201 [FWYILVM] ○○ DQA1*0501/ DQ7(3)[FYIMLV]-x-x-x-[VLIMY]-x-[YFMLVI] Marsh2000 24 DQB1*0301 ○ DQA1*0501/DQ7(3) [WYAVM]-x-x-[A]-x-[AIVTS]-x-x-x-[QN] SYFPEITHI 25 DQB1*0301 ○○○DQB1*0602 DQ6(1) [AFCILMNQSTVWYDE]-x-[AFGILMNQSTVWYCDE]- SYFPEITHI 68[AFGILMNQSTVWY]-x-[LIVAPST]-x-x-[ASTGLIVP] ○○○ DRB1*0101 DR1[YFWLIMVA]-x-x-[LMAIVN]-x-[AGSTCP]-x-x- Marsh2000 27 [LAIVNFYMW] ○○○DRB1*0101 DR1 [YVLFIAMW]-x-x-[LAIVMNQ]-x-[AGSTCP]-x-x- SYFPEITHI 28[LAIVNFY] ○○○ DRB1*0102 DR1 [ILVM]-x-x-[ALM]-x-[AGSTCP]-x-x-[ILAMYW]Marsh2000 29 ○○○ DRB1*0102 DR1 [ILVM]-x-x-[ALM]-x-[AGSTP]-x-x-[ILAMYW]SYFPEITHI 30 ○○ DRB1*0301 DR17(3)[LIFMV]-x-x-[D]-x-[KR(EQN)]-x-[L]-[YLF] Marsh2000 31 ○○ DRB1*0301DR17(3) [LIFMV]-x-x-[D]-x-[KREQN]-x-x-[YLF] SYFPEITHI 32 DRB1*0301 or DR17(3) [FILVY]-x-x-[DNQT] Marsh2000 33 DRB3*0201 DRB1*0301 or  DR17(3)unknown SYFPEITHI DRB3*0201 DRB1*0401 DR4 [FLV]-x-x-x-x-x-x-x-[NQST]Marsh2000 34 DRB1*0401 DR4 unknown SYFPEITHI ○○ DRB1*0401 or  DR4[FYWILVM]-x-x-[FWILVADE]-x-[NSTQHR]-x-x-[K] Marsh2000 35 DRB4DRB1*0401 or  DR4 [FYW]-x-x-x-x-x-x-x-[ST] Marsh2000 36 DRB4*0101 ○○○DRB1*0401 or  DR4 [FYWILVM]-x-x-[PWILVADE]-x-[NSTQHR]- SYFPEITHI 37DRB4*0101 [DEHKNQRSTYACILMV]-x-[DEHMQRSTYACILMV] ○○ DRB1*0402 or  DR4[VILM]-x-x-[YFWILMRNH]-x-[NSTQHK]-x- Marsh2000 38 DRB4 [RKHNQP]-x-[H]○○○ DRB1*0402 or  DR4 [VILM]-x-x-[YFWILMRN]-x-[NOTK]-[RKHNQP]-x-SYFPEITHI 39 DRB4 [DEHLWRSTYCILMVHA]

TABLE 4Candidate peptide sequences binding to various MHC class II molecules (WT1₁₈₆ peptides)Types of MHC  SEQ  class II Candidate peptide sequences binding ProgramID Suitability molecules Serotype to MHC class II molecules name NO: ○DRB1*0404 or  DR4 [VILM]-x-x[FYWILVMADE]-x-[NTSQR]-x-x-[K] Marsh2000 40DRB4 ○ DRB1*0404 or  DR4 [VILM]-x-x[FYWILVMADE]-x-[NTSQR]-x-x-[K]SYFPEITHI 40 DRB4 ○ DRB1*0405 or  DR4[FYWVILM1-x-x-[VILMDE]-x-[NSTQKD]-x-x-x-[DEQ] Marsh2000 41 DRB4 ○DRB1*0405 or  DR4 [FYWVILM]-x-x-IVILMDE]-x-[NSTQKD]-x-x-x-[DEQ]SYFPEITHI 41 DRB4 DRB1*0405 or  DR4 [Y]-x-x-x-x-[VT]-x-x-x-[D] Marsh200042 DRB4*0101 DRB1*0405 or  DR4 unknown SYFPEITHI DRB4*0101 DR31*0407 or DR4 [FYW]-x-x-[AVTK]-x-[NTDS]-x-x-x-[QN] Marsh2000 43 DRB4 DRB1*0407 or DR4 [FYW]-x-x-[AVK]-x-[NTDS]-x-x-x-[QN] SYFPEITHI 69 DRB4 DRB1*0701 DR7[FILVY]-x-x-x-x-[NST] Marsh2000 44 ○ DRB1*0701 DR7[FYWILV]-x-x-[DEHKNQRSTY]-x-[NST]-x-x-[VILYF] SYFPEITHI 70 DRB1*0801 DR8[FILVY]-x-x-x-[HKR] Marsh2000 46 DRB1*0801 DR8 unknown SYFPEITHIDRB1*0901 or  DR9 [YFWL]-x-x-[AS] Marsh2000 47 DRB4*0101 DRB1*0901 or DR9 [WYFL]-x-x-[AVS] SYFPEITHI 48 DRB4*0101 ○○ DRB1*1101 DR11(5)[YF]-x-x-[LVMAFY]-x-[RKH]-x-x-[AGSP] Marsh2000 49 ○○ DRB1*1101 DR11(5)[WYF]-x-x-[LVMAFY]-x-[RKH]-x-x-[AGSP] SYFPEITHI 71 DRB1 1101 or  DR11(5)[YF]-x-x-x-x-[RK]-x-[RK] Marsh2000 51 DRB3*0202 ○ DRB1*1104 DR11(5)[ILV]-x-x-[LVMAFY-x-[RKH]-x-x-[AGSP] Marsh2000 52 ○ DRB1*1104 DR11(5)[ILV]-x-x-[LVMAFY]-x-[RKH]-x-x-[AGSP] SYFPEITHI 52 DRB1*1201 or  DR12(5)[ILFY(V)]-x-[LNM(VA)]-x-x-[VY(FIN)]-x-x- Marsh2000 53 DRB3 [YFM(IV)] ○○DRB1*1201 or  DR12(5) [ILFYV]-x-LLMNVA]-x-x-[VYFINA]-x-x-[YFMIV]SYFPEITHI 54 DRB3 ○ DRB1*1301 DR13(6)[IVF]-x-x-[YWLVAM]-x-[RK]-x-x-[YFAST] Marsh2000 55 ○ DRB1*1301 DR13(6)[IVF]-x-x-[LVMAWY]-x-[RK]-x-x-[YFAST] SYFPEITHI 72 DRB1*1301 or  DR13(6)[ILV]-x-x-x-x-[RK]-x-x-[Y] Marsh2000 57 DRB3*0101 DRB1*1301 or  DR13(6)unknown SYFPEITHI DRB3*0101 ○ DRB1*1302 DR13(6)[YFVAI]-x-x-[YWLVAM]-x-[RK]-x-x-[YFAST] Marsh2000 58 ○ DRB1*1302 DR13(6)[YFVAI]-x-x-[LVMAWY]-x-[RK]-x-x-[YFAST] SYFPEITHI 59 DRB1*1302 or DR13(6) [ILFY]-x-x-x-x-[RK]-x-x-[Y] Marsh2000 60 DRB3*0301 DRB1*1302 or DR13(6) unknown SYFPEITHI DRB3*0301 ○ DRB1*1501 DR15(2)[LVI]-x-x-[FYI]-x-x-[ILVMF] Marsh2000 61 ○ DRB1*1501 DR15(2)[LVI]-x-x-[FYI]-x-x-[ILVMF] SYFPEITHI 61 ○ DRB1*1501 or  DR15(2)[ILV]-x-x-x-x-x-x-x-x-[HKR] Marsh2000 62 DRB5*0101 ○○○ DRB3*0202 DR52[YFIL]-x-x-[N]-x-[ASPDE]-x-x-[LVISG] Marsh2000 63 ○○○ DRB3*0202 DR52[YFIL]-x-x-[N]-x-[ASPDE]-x-x-[LVISG] SYFPEITHI 63 ○○○ DRB3*0301 DR52[ILV]-x-x-[N]-x-[ASPDE]-x-x-[ILV] Marsh2000 64 ○○○ DRB3*0301 DR52[ILV]-x-x-[N]-x-[ASPDE]-x-x-[ILV] SYFPEITHI 64 DRB5*0101 DR51[FYLM]-x-x-[QVIM]-x-x-x-x-[RK] Marsh2000 65 DRB5*0101 DR51[FYLM]-x-x-[QVIM]-x-x-x-x-[RK] SYFPEITHI 65

TABLE 5Candidate peptide sequences binding to various MHC class II molecules (WT1₂₉₄ peptides)Types of MHC  SEQ class II Candidate peptide sequences binding ProgramID  Suitability molecules Serotype to MHC class II molecules name NO:DPA1*0102/ DPw2 unknown Marsh2000 DPB1*0201 ○○ DPA1*0102/ DPw2[FLMVWY]-x-x-x-[FLMY]-x-x-[IAMV] SYFPEITHI  9 DPB1*0201 ○ DPA1*0103/DPw2 [FLM]-x-x-x-[FL]-x-x-[IA] Marsh2000 11 DPB1*0201 ○ DPA1*0103/ DPw2[YLVFK]-x-x-[DSQT]-x-[YFWV]-x-x-[LVI] Marsh2000 10 DPB1*0201 DPA1*0103/DPw2 unknown SYFPEITHI DPB1*0201 DPA1*0103/ DPw2 unknown SYFPEITHIDPB1*0201 ○○○ DPA1*0201/ DPw4 [FLYM(IVA)]-x-x-x-x-x-[FLY(MVIA)]-x-x-Marsh2000 12 DPB1*0401 [VYI(AL)] ○○○ DPA1*0201/ DPw4[FLYMIVA]-x-x-x-x-x-[FLYMVIA]-x-x-[VYIAL] SYFPEITHI 13 DPB1*0401DPA1*0201/ [RK]-x-x-x-x-[AGL]-x-x-[LV] Marsh2000 14 DPB1*0901 ○DPB1*0301 DPw3 x-[R]-x-x-x-x-x-x-x Marsh2000 15 DPB1*0301 DPw3 unknownSYFPEITHI DQA1*0101/ DQ5(1) [L]-x-x-x-[YFW] Marsh2000 16 DQB1*0501DQA1*0101/ DQ5(1) unknown SYFPEITHI DQB1*0501 DQA1*0102/ DQ6(1)x-x-x-x-x-[LIV(APST)]-x-x-[AGST(LIVP)] Marsh2000 17 DQB1*0602 DQA1*0301/DQ7(3) x-x-[AGST]-x-[AVLI] Marsh2000 18 DQB1*0301 DQA1*0301/ DQ7(3)[DEW]-x-x-[AGST]-x-[ACLM] SYFPEITHI 19 DQB1*0301 DQA1*0301/ DQ8(3)[RK]-x-x-x-x-[AG]-x-x-[NED] Marsh2000 66 DQB1*0302 ○ DQA1*0301/ DQ8(3)[TSW]-x-x-x-x-x-x-x-[RE] SYFPEITHI 21 DQB1*0302 ○○○ DQA1*0501/ DQ2[FWYILV]-x-x-[DELVIH]-x-[PDE(H)]-[ED]-x- Marsh2000 22 DQB1*0201[FYWVILM] ○○○ DQA1*0501/ DQ2 [FWYILV]-x-x-[DELVIH]-x-[PDEHPA]-[DE]-x-SYFPEITHI 67 DQB1*0201 [FWYILVM] ○○○ DQA1*0501/ DQ7(3)[FYIKLV]-x-x-x-[VLIMY]-x-[YFMLVI] Marsh2000 24 DQB1*0301 DQA1*0501/DQ7(3) [WYAVM]-x-x-[A[-x-[AIVTS]-x-x-x-[QN] SYFPEITHI 25 DQB1*0301 ○○○DQB1*0602 DQ6(1) [AFCILMNQSTVWYDE]-x-[AFGILMNQSTVWYCDE]- SYFPEITHI 68[AFGILMNQSTVWY]-x-[LIVAPST]-x-x-[ASTGLIVP] ○○○ DRB1*0101 DR1[YFWLIMVA]-x-x-[LMAIVN]-x-[AGSTCP]-x-x- Marsh2000 27 [LAIVNFYMW] ○○○DRB1*0101 DR1 [YVLFIAMW]-x-x-[LAIVMNQ]-x-[AGSTCP]-x-x- SYFPEITHI 28[LAIVNFY] DRB1*0102 DR1 [ILVM]-x-x-[ALM]-x-[AGSTCP]-x-x-[ILAMYW]Marsh2000 29 DRB1*0102 DR1 [ILVM]-x-x-[ALM]-x-[AGSTP]-x-x-[ILAMYW]SYFPEITHI 30 ○○ DRB1*0301 DQ17(3) [LIFMV]-x-x-[D]-x-[KR(EQN)]-x-[L]-YLF]Marsh2000 ○○ DRB1*0301 DQ17(3) [LIFMV]-x-x-[D[-x-[KREQN]-x-x-[YLF]SYFPEITHI 32 ○○ DRB1*0301 or DQ17(3) [FILVY]-x-x-[DNQT] Marsh2000 33DRB3*0201 DRB1*0301 or DQ17(3) unknown SYFPEITHI DRB3*0201 ○○ DRB1*0401DR4 [FLV]-x-x-x-x-x-x-x-[NQST] Marsh2000 34 DRB1*0401 DR4 unknownSYFPEITHI ○○○ DRB1*0401 or  DR4[FYWILVM]-x-x-[FWILVADE]-x-[NSTQHR]-x-x-[K] Marsh2000 35 DRB4 ○○DRB1*0401 or DR4 [FYW]-x-x-x-x-x-x-x-[ST] Marsh2000 36 DRB4*0101 ○○○DRB1*0401 or DR4 [FYWILVM]-x-x-[PWILVADEl-x-[NSTQHR]- SYFPEITHI 37DRB4*0101 [DEHKNQRSTYACILMV]-x-[DEHKNQRSTYACILMV] + ○○○ DRB1*0402 or DR4 [VILM]-x-x-[YFWILMRNH]-x-[NSTQHK]-x-[RKHNQP]- Marsh2000 38 DRB4x-[H] ○○○ DRB1*0402 or  DR4 [VILM]-x-x-[YFWILMRN]-x-[NQSTK]-[RKHNQP]-x-SYFPEITHI 39 DRB4 [DEHLNQRSTYCILMVHA]

TABLE 6Candidate peptide sequences binding to various MHC class II molecules (WT1₂₉₄ peptides)Types of MHC  SEQ  class II Candidate peptide sequences binding  ProgramID Suitability molecules Serotype to MHC class II molecules name NO: ○DRB1*0404 or  DR4 [VILM]-x-x-[FYWILVMADE]-x-[NTSQN-x-x-[K] Marsh2000 40DRB4 ○ DRB1*0404 or  DR4 [VILM]-x-x-[FYWILVMADE]-x-[NTSQR]-x-x-[K]SYFPEITHI 40 DRB4 ○○○ DRB1*0405 or  DR4[FYWVILM]-x-x-[VILMDE]-x-[NSTQKD]-x-x-x- Marsh2000 41 DRB4 [DEQ] ○○○DRB1*0405 or  DR4 [FYWVILM]-x-x-[VILMDE]-x-[NSTQKD]-x-x-x- SYFPEITHI 41DRB4 [DEQ] DRB1*0405 or  DR4 [Y]-x-x-x-x-[VT]-x-x-x-[D] Marsh2000 42DRB4*0101 DRB1*0405 or  DR4 unknown SYFPEITHI DRB4*0101 ○○○DRB1*0407 or  DR4 [FYW]-x-x-[-AVTK]-x-[NTDS]-x-x-x-[QN] Marsh2000 43DRB4 ○○○ DRB1*0407 or  DR4 [FYW]-x-x-[AVK]-x-[NTDS]-x-x-x-[QN] SYFPEITHI69 DRB4 ○ DRB1*0701 DR7 [FILVY]-x-x-x-x-[NST] Marsh2000 44 ○ DRB1*0701DR7 [FYWILV]-x-x-[DEHKNQRSTY]-x-[NST]-x-x- SYFPEITHI 70 [VILYF] ○DRB1*0801 DRB [FILVY]-x-x-x-[HKR] Marsh2000 46 DRB1*0801 DRB unknownSYFPEITHI ○ DRB1*0901 or  DR9 [YFWL]-x-x-[AS] Marsh2000 47 DRB4*0101 ○DRB1*0901 or  DR9 [WYFL]-x-x-[AVS] SYFPEITHI 48 DRB4*0101 ○○ DRB1*1101DR11(5) [YF]-x-x-[LVMAFY]-x-[RKH]-x-x-[AGSP] Marsh2000 49 ○○ DRB1*1101DR11(5) [WYF]-x-x-[LVMAFY]-x-[RKH]-x-x-[AGSP] SYFPEITHI 71 ○○○DRB1*1101 or  DR11(5) [YF]-x-x-x-x-[RK]-x-[RK] Marsh2000 51 DRB3*0202DRB1*1104 DR11(5) [ILV]-x-x-[LVMAFY]-x-[RKH]-x-x-[AGSP] Marsh2000 52DRB1*1104 DR11(5) [ILV]-x-x-[LVMAFY]-x-[RKH]-x-x-[AGSP] SYFPEITHI 52 ○○DRB1*1201 or  DR12(5) [ILFY(V)]-x-[LNM(VA)]-x-x-[VY(FIN)]-x-x- Marsh200053 DRB3 [YFM(IV)] ○○ DRB1*1201 or  DR12(5)[ILFYV]-x-[LMNVA]-x-x-[VYFINA]-x-x-[YFMIV] SYFPEITHI 54 DRB3 DRB1*1301DR13(6) [IVF]-x-x-[YWLVAM]-x-[RK]-x-x-[YFAST] Marsh2000 55 DRB1*1301DR13(6) [ILV]-x-x-[LVMAWY]-x-[RK]-x-x-[YFAST] SYFPEITHI 56 DRB1*1301 or DR13(6) [ILV]-x-x-x-x-[RK]-x-x-[Y] Marsh2000 57 DRB3*0101 DRB1*1301 or DR13(6) unknown SYFPEITHI DRB3*0101 ○ DRB1*1302 DR13(6)[YFVAI]-x-x-[YWLVAM]-x-[RK]-x-x-[YEAST] Marsh2000 73 ○ DRB1*1302 DR13(6)[YFVAI]-x-x-[LVMAWY]-x-[RK]-x-x-[YEAST] SYFPEITHI 74 ○ DRB1*1302 or DR13(6) [ILFY]-x-x-x-x-[RK]-x-x-[Y] Marsh2000 60 DRB3*0301 DRB1*1302 or DR13(6) unknown SYFPEITHI DRB3*0301 ○○ DRB1*1501 DR15(2)[LVI]-x-x-[FYI]-x-x-[ILVMF] Marsh2000 61 ○○ DRB1*1501 DR15(2)[LVI]-x-x-[FYI]-x-x-[ILVMF] SYFPEITHI 61 DRB1*1501 or  DR15(2)[ILV]-x-x-x-x-x-x-x-x-[HKR] Marsh2000 62 DRB5*0101 ○○○ DRB3*0202 DR52[YFIL]-x-x-[N]-x-[ASPDE]-x-x-[LVISG] Marsh2000 63 ○○○ DRB3*0202 DR52[YFIL]-x-x-[N]-x-[ASPDE]-x-x-[LVISG] SYFPEITHI 63 ○ DRB3*0301 DR52[ILV]-x-x-[N]-x-[ASPDE]-x-x-[ILV] Marsh2000 64 ○ DRB3*0301 DR52[ILV]-x-x-[N]-x-[ASPDE]-x-x-[ILV) SYFPEITHI 64 ○○○ DRB5*0101 DR51[FYLM]-x-x-[QVIM]-x-x-x-x-[RK] Marsh2000 65 ○○○ DRB5*0101 DR51[FYLM]-x-x-[QVIM]x x x x[RK] SYFPEITHI 65

Next, candidate WT1 peptides were visually selected from Tables 1 to 6,peptides as shown in the following Table 7 were identified as preferredcandidate peptides for MHC class II molecules, and actual functions ofthese peptides were analyzed as described below.

TABLE 7 Identification of peptide candidates formouse MHC class II molecules WT1₃₅ WAPVLDFAPPGASAYGSL 18 mer MW 1819.01(SEQ ID NO: 3) WT1₈₆ EQCLSAFTLHFSGQFTG 17 mer MW 1944.01 (SEQ ID NO: 6)WT1₂₉₄ FRGIQDVRRVSGVAPTLVR 19 mer MW 2126.48 (SEQ ID NO: 7)

Preparation of WT1 Peptide-Specific Cell Lines and Measurement of CellProliferation Ability

First, the above WT1 peptides were emulsified with a Freund's incompleteadjuvant (Montanide ISA 51), and mice were intradermally inoculated witheach WT1 peptide in an amount corresponding to 100 μg/mouse. Theimmunization was carried out 3 times at intervals of one week, thespleen was removed after 1 week of the final immunization, and spleencells were prepared. The spleen cells were stimulated 3 times atintervals of 10 days using spleen cells of non-immunized mice, whichwere pulsed with the same WT1 peptide as that used for immunization ofeach mouse and irradiated, as a stimulator. Then, the 4th stimulationwas carried out using spleen cells of non-immunized mice, which werepulsed with each peptide (WT1₃ ₅, WT1₈ ₆ or WT1₂ ₉ ₄ peptide) as shownin Table 7 and irradiated, as a stimulator, and proliferation reactionin response to each stimulator was measured by a ³H incorporationexperiment. An OVA (ovalbumin) peptide irrelevant to WT1 peptides wasused as a control peptide. As a result, mouse spleen cells immunizedwith a WT1₃ ₅ peptide, a WT1₈ ₆ peptide or a WT1₂ ₉ ₄ peptide eachresponded to the stimulator pulsed with a WT1₃ ₅ peptide, a WT1₈ ₆peptide or a WT1₂ ₉ ₄ peptide, and proliferated (FIG. 1A to 1C).

As described above, spleen cells were stimulated in vitro 3 times atintervals of 10 days using spleen cells of non-immunized mice, whichwere pulsed with each WT1 peptide and irradiated. When the 4thstimulation was then carried out using spleen cells of non-immunizedmice, which were pulsed with each peptide described above andirradiated, as a stimulator, and proliferation reaction was measured, anMHC class I antibody (D^(b) antibody) or an MHC class II antibody (Abantibody) was added to the culture solution and ³H incorporation wasmeasured. As a result, the proliferation reaction in response to thestimulator pulsed with each of a WT1₃ ₅ peptide, a WT1₈ ₆ peptide and aWT1₂ ₉ ₄ peptide was suppressed by the addition of an MHC class IIantibody (FIG. 2A to 2C).

As described above, spleen cells were stimulated in vitro 3 times atintervals of 10 days using spleen cells of non-immunized mice, whichwere pulsed with each WT1 peptide and irradiated. Then, theproliferation reaction was measured by ³H incorporation using irradiatedC1498 cells not expressing any WT1 protein, C1498 cells pulsed with eachof the above WT1 peptides, or C1498 cells expressing a WT1 protein byintroduction of a WT1 gene, as a stimulator.

As a result, the proliferation reaction was produced in response toC1498 cells pulsed with the same WT1 peptide as that used inimmunization in vivo and C1498 cells expressing a WT1 protein byintroduction of a WT1 gene (FIG. 3 ). This revealed that a WT1₃ ₅peptide, a WT1₈ ₆ peptide and a WT1₂ ₉ ₄ peptide are produced by anintracellular process of an endogenous WT1 protein and displayed on anMHC class II molecule. From the above facts, it was shown that thesethree WT1 peptides are MHC class II-restricted WT1 peptides.

Measurement of IFN-γ Producing Ability

As described above, spleen cells were stimulated in vitro 3 times atintervals of 10 days using spleen cells of non-immunized mice, whichwere pulsed with each WT1 peptide and irradiated. Then, theconcentration of IFN-γ and IL-4 in a culture supernatant was measuredusing an ELISA kit (BIOSOURCE Immunoassay Kit, Invitrogen). As a result,spleen cells of two separate mice responded to spleen cells ofnon-immunized mice which were pulsed with each WT1 peptide andirradiated, and produced interferon-γ but little interleukin-4 (FIG. 4). This revealed that these three types of WT1 peptides induce Th1 typeof WT1-specific helper T cells.

Example 2

Measurement of WT1-Specific Cytotoxic T Cells (CTLs)

Mice were immunized 3 times with a WT1₁ ₂ ₆ peptide (MHC class I) alone,a WT1₁ ₂ ₆ peptide (MHC class I)+a WT1₃ ₅ peptide (MHC class II), a WT1₁₂ ₆ peptide (MHC class I)+a WT1₈ ₆ peptide (MHC class II), or a WT1₁ ₂ ₆peptide (MHC class I)+a WT1₂ ₉ ₄ peptide (MHC class II), and spleencells of the mice were prepared. Then, the spleen cells were stimulatedonce in vitro using a WT1₁ ₂ ₆ peptide (MHC class I), and on 6th day,cytotoxic activity was measured using RMAS cells pulsed with a WT1₁ ₂ ₆peptide (MHC class I) as a target cell. RMAS cells not pulsed with aWT1₁ ₂ ₆ peptide (MHC class I) were used as a control target cell. As aresult, mouse spleen cells immunized with a WT1₁ ₂ ₆ peptide (MHC classI)+a WT1 helper peptide (MHC class II) induced WT1-specific cytotoxic Tcells more strongly as compared with mouse spleen cells immunized with aWT1₁ ₂ ₆ peptide (MHCI class I) alone (FIG. 5 ). This demonstrated thatthe three WT1 peptides (MHC class II) are a WT1-specific helperpeptides.

Example 3

Tumor Implantation Experiment

WT1-expressing C1498 leukemia cells were subcutaneously implanted inmice in a proportion of 2.5×10⁵ cells per mouse, and 50 μg/mouse of aWT1₃ ₅ helper peptide was intradermally administered together with aFreund's incomplete adjuvant, once a week, 3 times in total, startingfrom one week after the implantation (FIG. 6 ). As a control, aphysiological saline instead of the WT1₃ ₅ helper peptide wasintradermally administered together with a Freund's incomplete adjuvant.The size of a subcutaneous tumor was measured over time, and thedisease-free survival rate was calculated up to the 29th day after thesubcutaneous implantation. As a result, the tumor expanded in all miceof the control group, while proliferation of the tumor was completelysuppressed in 4 of 10 mice of the WT1₃ ₅ helper peptide (MHC classII)-immunized group (FIG. 7). Also, a significant difference (p<0.05)was recognized between the WT1₃ ₅ helper peptide-immunized group and thecontrol group (FIG. 8 ). This demonstrated that the WT1₃ ₅ helperpeptide (MHC class II) is a WT1 peptide having an ability to inducetumor immunization in vivo.

Next, mice were dissected on the 29th day after starting the aboveexperiment, the spleen was excised, and a WT1-specific immune responsewas analyzed using spleen cells. Briefly, the spleen was excised whenmice of the WT1₃ ₅ helper peptide (MHC class II)-immunized group and thecontrol group were dissected, and spleen cells were prepared. The spleencells were stimulated once with a WT1₁ ₂ ₆ peptide (MHC class I), and onthe 6th day after the stimulation, cytotoxic activity of the spleencells was measured using RMAS cells pulsed with a WT1₁ ₂ ₆ peptide (MHCclass I) as a target cell. As a control, the cytotoxic activity of thespleen cells was measured using RMAS cells as a target cell. As aresult, WT1-specific cytotoxic T cells were induced in all 4 mice of theWT1₃ ₅ helper peptide (MHC class II)-immunized group (FIG. 9 ). On theother hand, the WT1-specific cytotoxic T cells were very weakly inducedin 3 mice of the control group (FIG. 10 ). The WT1-specific cytotoxic Tcells were not induced in one mouse. Also, it was clear that theinduction of the WT1-specific cytotoxic T cells was lower as comparedwith the WT1₃ ₅ helper peptide (MHC class II)-immunized group (FIGS. 9and 10 ). This shows that WT1-specific helper T cells were induced byadministration of a WT1₃ ₅ class II helper peptide, and by the action ofthe WT1-specific helper T cells, WT1-specific cytotoxic T cells inducedby immune-responding to a WT1 protein expressed by implanted tumor cellswere strongly amplified in vivo. Thus, the results demonstrated theusefulness of the WT1₃ ₅ helper peptide.

Next, specific cytolysis was analyzed in mice of the above WT1₃₅ helperpeptide (MHC class II)-immunized group and control group. Briefly, thedegree of cytolysis (%) obtained by subtracting the rate of cytolysis(%) when target cells were RMAS cells from the rate of cytolysis (%)when target cells were RMAS cells pulsed with a WT1₁₂₆ peptide (MHCclass I) in the above experiments was used as the specific cytolysis (%)(FIG. 11 , left). Also, the above-prepared spleen cells and afluorescence-labeled WT1 tetramer (H-2 Db WT1 Tetramer-RMFPNAPYL-PE)were incubated at 4° C. for 20 minutes, washed, then stained withfluorescence-labeled CD3 and CD8 antibodies, again washed, and analyzedby FACS. CD3-positive, CD8-positive, and WT1 tetramer-positive cellswere served as WT1-specific cytotoxic T cells (FIG. 11 , right). As aresult, significantly high WT1-specific cytotoxic T cells (p<0.05) wereinduced in spleen cells of mice of the WT1₃₅ helper peptide (MHC classII)-immunized group as compared with spleen cells of mice of the controlgroup (FIG. 11 ).

Example 4

Measurement of Proliferation Ability of WT1-Specific Cytotoxic T Cells(CTLs) in Human

Peripheral blood mononuclear cells were prepared from 6 healthy subjectshaving DRB1, DPB1, DQB1 or DRB5 subclass molecules as shown in FIG. 12 .To the peripheral blood mononuclear cells, a WT1₃ ₅ helper peptide wasadded, and the cells were cultured for one week. Then, the peripheralblood mononuclear cells were stimulated 4 times in total at intervals ofone week using identical subject-derived peripheral blood mononuclearcells, which were pulsed with a WT1₃ ₅ helper peptide and irradiated, asa stimulator, and ³H incorporation was measured on the 6th day. In all 6healthy subjects, peripheral blood mononuclear cells responded to a WT1₃₅ helper peptide and proliferated (FIG. 12 ). This showed that the WT1₃₅ helper peptide has a function to bind to the mentioned HLA class IImolecules and cause proliferation reaction. In this connection, themouse WT1₈ ₆ peptide and WT1₂ ₉ ₄ peptide differ from the human WT1₆peptide (SEQ ID NO:4) and WT1₂ ₉ ₄ peptide (SEQ ID NO:5) in one aminoacid at the positions enclosed in squares, as shown in Table 8.

TABLE 8 Differences in sequences between mouse andhuman WT1₃₅, WT1₈₆ and WT1₂₉₄ peptides mWT1₃₅ Mouse WAPVLDFAPPGASAYGSL18-mer (SEQ ID NO: 3) hWT1₃₅ Human WAPVLDFAPPGASAYGSL (SEQ ID NO: 3)mWT1₈₆ Mouse EQCLSAFTLHFSGQFTG 17-mer (SEQ ID NO: 6) hWT1₈₆ HumanEQCLSAFTVHFSGQFTG (SEQ ID NO: 4) mWT1₂₉₄ Mouse FRGIQDVRPVSGVAPTLVR19-mer (SEQ ID NO: 7) hWT1₂₉₄ Human FRGIQDVRRVPGVAPTLVR (SEQ ID NO: 5)

Example 5

HLA Class II Molecule-Restrictedness of WT1₃ ₅ Peptide

In order to determine HLA class II molecule-restrictedness of a WT1₃ ₅peptide, a further experiment was carried out by a method well known tothose skilled in the art as briefly described below. First, peripheralblood mononuclear cells (PBMCs) derived from a healthy subject [aDRB1*0101/0405-, DPB1*0201/0402-, and DQB1*0401/0501-positive healthysubject (hereinafter referred to as healthy subject A)] were stimulated5 times with a WT1₃ ₅ peptide to prepare a Responder. Next, peripheralblood mononuclear cells (PBMCs) derived from another healthy subjectdifferent in an HLA class II type [a DRB1*0405/0901-, DPB1*0201/0501-,and DQB1*0303/0401-positive healthy subject (referred to as healthysubject B)] were pulsed with the WT1₃ ₅ peptide to prepare a Stimulator,and cell proliferation [the amount of ³H-thymidine incorporated (cpm)]was measured. The measurement was carried out under conditions of noaddition of an antibody, addition of an anti-HLA-DR antibody (+a-DR),addition of an anti-HLA-DP antibody (+a-DP), or addition of ananti-HLA-DQ antibody (+a-DQ). A common HLA class II type, which ispositive in both the Responder and Stimulator, shows restrictedness ofthe WT1₃ ₅ peptide. As a result of the experiments, it was shown thatthe WT1₃ ₅ peptide is DRB1*0405-restricted because the proliferation wassuppressed under a condition having addition of an anti-DR antibody, andDRB10405 was common in healthy subjects A and B, as shown in FIG. 13 .

Next, an experiment was carried out under the same conditions as thoseof the above experiment, except that PBMCs derived from a healthysubject different from healthy subject A [DRB1*0405/0803-,DPB1*0202/0501-, and DQB1*0401/0601-positive healthy subject (referredto as healthy subject G)] were used as a Stimulator. As a result, it wasshown that the WT1₃ ₅ peptide is DRB1*0405-, DPB1*0201- andDPB1*0202-restricted because the proliferation was suppressed under acondition having addition of an anti-HLA-DR antibody or an anti-HLA-DPantibody, and DRB1*0405, DPB1*0201 and DPB1*0202 were common in healthysubject A and healthy subject G (DPB1*0201 and DPB1*0202 have a highanalogy and are cross-reactive, and therefore, they are considered as acommon molecule), as shown in FIG. 14 .

Next, an experiment was carried out under the same conditions as thoseof the above experiment, except that PBMCs derived from a healthysubject different from healthy subject A [DRB1*0101/0803, DPB1*0501/-,DQB1*0501/0601-positive (referred to as healthy subject H)] were used asa Stimulator. As a result, it was shown that the WT1₃ ₅ peptide isDRB1*0101-restricted because the proliferation was suppressed under acondition having addition of an anti-HLA-DR antibody, and DRB1*0101 wascommon in healthy subject A and healthy subject H, as shown in FIG. 15 .

Moreover, PBMCs derived from healthy subject G were used as a Responderand L cells having a DQB1*0601 gene introduced were used as aStimulator, in order to determine restrictedness of a WT1₃ ₅ peptide.The difference in an amount of IFN-γ produced in the presence or absenceof a pulse with a WT1₃ ₅ peptide of L cells was measured. A proportionof intracellular IFN-γ production was measured using FACS which is atechnique well known to those skilled in the art. As a result, it wasshown that the WT1₃ ₅ peptide is DQB1*0601-restricted because theResponder was activated by the pulse with a WT1₃ ₅ peptide on L cells,as shown in FIG. 16 .

Next, an experiment was carried out as described above using PBMCsderived from the same healthy subject as a Responder and a Stimulator.The types of HLA class II molecules possessed by healthy subjects usedin this experiment were summarized in Table 9 below.

TABLE 9 Types of HLA class II molecules possessed by healthy subjectsused in this experiment Healthy subject No. DRB1 DPB1 DQB1 A *0101/0405*0201/0402 *0401/0501 B *0405/0901 *0201/0501 *0303/0401 C *0802/1201*0201/0501 *0301/0302 D *1502/1502 *0201/0901 *0601/0601 E *0405/0901*0202/0501 *0303/0401 F *1403/1502 *0201/0901 *0301/0601 G *0405/0803*0202/0501 *0401/0601 H *0101/0803 *0501/— *0501/0601 I *0101/1501*0201/0402 *0501/0602

As a result, it was found that addition of an anti-DR antibody or ananti-DP antibody, when the experiment was carried out using PBMCsderived from healthy subjects A to E, resulted in reduction of theamount of ³H-thymidine incorporated (cpm), and therefore, in suppressionof the proliferation. Also, addition of only an anti-DR antibody, whenPBMCs derived from healthy subject F were used, resulted in suppressionof the proliferation. Moreover, addition of only an anti-HLA-DPantibody, when PBMCs derived from healthy subject G were used, resultedin suppression of the proliferation. By an experiment using healthysubject A, it was shown that the WT1₃ ₅ peptide is DRB1*0101- or0405-restricted, and DPB1*0201- or 0402-restricted. By an experimentusing healthy subject B, it was shown that the WT1₃ ₅ peptide isDRB1*0405- or 0901-restricted, and DPB1*0201- or 0501-restricted. By anexperiment using healthy subject C, it was shown that the WT1₃ ₅ peptideis DRB1*0802- or 1201-restricted, and DPB1*0201- or 0501-restricted. Byan experiment using healthy subject D, it was shown that the WT1₃ ₅peptide is DRB1*1502-restricted because the DRB1*1502 is a homozygote(FIG. 17 ). In addition, it was shown that the WT1₃ ₅ peptide isDPB1*0201- or 0901-restricted. By an experiment using healthy subject E,it was shown that the WT1₃ ₅ peptide is DRB1*0405- or 0901-restricted,and DPB1*0202- or 0501-restricted. By an experiment using healthysubject F, it was shown that the WT1₃₅ peptide is DRB1*1403- or1502-restricted. By an experiment using healthy subject G, it was shownthat the WT1₃ ₅ peptide is DPB1*0202- or 0501-restricted.

Also, the difference in an amount of IFN-γ produced in the presence orabsence of a pulse with a WT1₃ ₅ peptide was measured using PBMCsderived from healthy subject I as a Responder and a Stimulator. Aproportion of intracellular IFN-γ production was measured using FACSwhich is a technique well known to those skilled in the art. As aresult, a proportion of an amount of IFN-γ remarkably increased by thepulse with a WT1₃ ₅ peptide (FIG. 18 ). This shows that the WT1₃ ₅peptide is restricted by any one of DRB1*0101, DRB1*1501, DPB1*0201,DPB1*0402, DQB1*0501, and DQB1*0602.

INDUSTRIAL APPLICABILITY

The present invention provides a WT1 peptide which is restricted by manytypes of MHC class II molecules, a polynucleotide encoding the peptide,a pharmaceutical composition containing them and the like. Thus, theycan be utilized in the field of pharmaceuticals, for example, the fieldof the development and production of prophylactic or therapeutic drugsfor various hematopoietic organ tumors and solid tumors which highlyexpress a WT1 gene.

[Sequence Listing Free Text]

The invention claimed is:
 1. A peptide that induces WT1-specific helperT cells by binding to an MHC class II molecule, wherein the peptideconsists of an amino acid sequence in which one to three amino acids aresubstituted in the amino acid sequence depicted in SEQ ID NO:5.
 2. Thepeptide according to claim 1, wherein the MHC class II molecule isselected from the group consisting of DRB1*0101, DRB1*0405 DRB1*0802,DRB1*0803, DRB1*0901, DRB1*1201, DRB1*1403, DRB1*1501, DRB1*1502,DPB1*0201, DPB1*0202, DPB1*0402, DPB1*0501, DPB1*0901, DQB1*0301,DQB1*0302, DQB1*0401, DQB1*0501, DQB1*0601, DQB1*0602 and DRB5*0102. 3.The peptide according to claim 1, wherein the MHC class II molecule isselected from the group consisting of DRB1*0101, DRB1*0405 DRB1*1502,DPB1*0201, DPB1*0202 and DQB1*0601.
 4. A pharmaceutical composition fortreating or preventing cancer, comprising the peptide according toclaim
 1. 5. A method for treating or preventing cancer, which comprisesadministering an effective amount of the peptide according to claim 1 toa subject having an MHC class II molecule selected from the groupconsisting of DRB1*0101, DRB1*0405, DRB1*0802, DRB1*0803, DRB1*0901,DRB1*1201, DRB1*1403, DRB1*1501, DRB1*1502, DPB1*0201, DPB1*0202,DPB1*0402 DPB1*0501, DPB1*0901, DQB1*0301, DQB1*0302 DQB1*0401,DQB1*0501, DQB1*0601, DQB1*0602, and DRB5*0102.
 6. Antigen presentingcells which display the peptide according to claim 1 through an MHCclass II molecule selected from the group consisting of DRB1*0101,DRB1*0405, DRB1*0802, DRB1*0803, DRB1*0901, DRB1*1201, DRB1*1403,DRB1*1501, DRB1*1502, DPB1*0201, DPB1*0202, DPB1*0402, DPB1*0501,DPB1*0901, DQB1*0301, DQB1*0302, DQB1*0401, DQB1*0501, DQB1*0601,DQB1*0602, and DRB5*0102.
 7. A method for inducing antigen presentingcells, which comprises culturing immature antigen presenting cells inthe presence of the peptide according to claim 1, and inducing antigenpresenting cells, which display the peptide through an MHC class IImolecule selected from the group consisting of DRB10101, DRB1*0405,DRB1*0802, DRB1*0803, DRB1*0901, DRB1*1201, DRB1*1403, DRB1*1501,DRB1*1502, DPB1*0201, DPB1*0202, DPB1*0402, DPB10501, DPB1*0901,DQB1*0301, DQB1*0302, DQB1*0401, DQB1*0501, DQB1*0601, DQB1*0602, andDRB5*0102, from the immature antigen presenting cells.
 8. WT1-specifichelper T cells which are induced by the peptide according to claim
 1. 9.A method for inducing WT1-specific helper T cells, which comprisesculturing peripheral blood mononuclear cells in the presence of thepeptide according to claim 1, and inducing WT1-specific helper T cellsfrom the peripheral blood mononuclear cells.
 10. A kit for inducingWT1-specific helper T cells, comprising, as an essential ingredient, thepeptide according to claim
 1. 11. A kit for preventing or treatingcancer, comprising, as an essential ingredient the peptide according toclaim
 1. 12. A method for determining the presence or amount ofWT1-specific helper T cells in a subject having an MHC class II moleculewhich is selected from the group consisting of DRB1*0101, DRB1*0405,DRB1*0802, DRB1*0803, DRB1*0901, DRB1*1201, DRB1*1403, DRB1*1501,DRB1*1502, DPB1*0201, DPB1*0202, DPB1*0402, DPB1*0501, DPB1*0901,DQB1*0301, DQB1*0302, DQB1*0401, DQB1*0501, DQB1*0601, DQB1*0602, andDRB5*0102, wherein said method comprises the steps of: (a) reacting thepeptide according to claim 1 with a sample derived from the subject; and(b) determining the presence or amount of a cytokine contained in thesample.